Methods of identifying a composition that alters connective tissue growth factor expression

ABSTRACT

The present invention provides a novel polypeptide, Connective Tissue Growth Factor (CTGF), polynucleotides encoding CTGF, and including 5&#39; and 3&#39; untranslated nucleotides, antibodies reactive with CTGF and means for producing CTGF. Also provided are diagnostic and therapeutic methods for using CTGF, as well as an assay for identifying compositions which affect the expression of CTGF polynucleotide. The invention provides a novel TGF-β responsive element upstream of the polynucleotide encoding CTGF structural gene.

This invention was made with Government support by grant no. GM 37223,awarded by the National Institutes of Health. The Government has certainrights in this invention.

This is a divisional of U.S. patent application Ser. No. 08/459,717,filed Jun. 2, 1995, now issued as U.S. Pat. No. 5,770,207, which is acontinuation-in-part application of Ser. No. 08/386,680, filed Feb. 10,1995, now issued as U.S. Pat. No. 5,585,270, which is a divisional ofSer. No. 08/167,628, filed on Dec. 14, 1993, now issued as U.S. Pat. No.5,408,040, which is a continuation of Ser. No. 07/752,427, filed Aug.30, 1991, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the field of growth factors andspecifically to Connective Tissue Growth Factor (CTGF), a polynucleotideencoding this factor and methods of use for CTGF.

2. Related Art

Growth factors are a class of secreted polypeptides that stimulatetarget cells to proliferate, differentiate and organize in developingtissues. The action of growth factors is dependent on their binding tospecific receptors which stimulates a signalling event within the cell.Examples of some well-studied growth factors include platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF-I), transforminggrowth factor beta (TGF-β), transforming growth factor alpha (TGF-α),epidermal growth factor (EGF), and fibroblast growth factor (FGF).

PDGF is a cationic, heat-stable protein found in the alpha-granules ofcirculating platelets and is known to be a mitogen and a chemotacticagent for connective tissue cells such as fibroblasts and smooth musclecells. Because of the activities of this molecule, PDGF is believed tobe a major factor involved in the normal healing of wounds andpathologically contributing to such diseases as atherosclerosis andfibrotic diseases. PDGF is a dimeric molecule consisting of an A chainand a B chain. The chains form heterodimers or homodimers and allcombinations isolated to date are biologically active.

Studies on the role of various growth factors in tissue regeneration andrepair have led to the discovery of PDGF-like proteins. These proteinsshare both immunological and biological activities with PDGF and can beblocked with antibodies specific to PDGF.

These new growth factors may play a significant role in the normaldevelopment, growth, and repair of human tissue. Therapeutic agentsderived from these molecules may be useful in augmenting normal orimpaired growth processes involving connective tissues in certainclinical states, e.g., wound healing. When these growth factors areinvolved pathologically in diseases, therapeutic developments from theseproteins may be used to control or ameliorate uncontrolled tissuegrowth.

The formation of new and regenerating tissue requires the coordinateregulation of various genes that produce both regulatory and structuralmolecules which participate in the process of cell growth and tissueorganization. Transforming growth factor beta (TGF-β) appears to be acentral regulatory component of this process. TGF-β is released byplatelets, macrophages and neutrophils which are present in the initialphases of the repair process. TGF-β can act as a growth stimulatoryfactor for mesenchymal cells and as a growth inhibitory factor forendothelial and epithelial cells. The growth stimulatory action of TGF-βappears to be mediated via an indirect mechanism involving autocrinegrowth factors such as PDGF BB or PDGF AA or connective tissue growthfactor (CTGF).

Several members of the TGF-β superfamily possess activities suggestingpossible applications for the treatment of cell proliferative disorders,such as cancer. In particular, TGF-β has been shown to be potent growthinhibitor for a variety of cell types (Massague, Cell 49:437, 1987), MIShas been shown to inhibit the growth of human endometrial carcinomatumors in nude mice (Donahoe, et al., Ann. Surg. 194:472, 1981), andinhibin α has been shown to suppress the development of tumors both inthe ovary and in the testis (Matzuk, et al., Nature, 360:313, 1992).

Many of the members of the TGF-β family are also important mediators oftissue repair. TGF-β has been shown to have marked effects on theformation of collagen and causes of striking angiogenic response in thenewborn mouse (Roberts, et al., Proc. Natl. Acad. Sci., USA83:4167,1986). The bone morphogenic proteins (BMPs) can induce new bonegrowth and are effective for the treatment of fractures and otherskeletal defects (Glowacki, et al., Lancet, 1:959, 1981; Ferguson, etal., Clin. Orthoped. Relat. Res., 227:265, 1988; Johnson, et al., ClinOrthoped. Relat. Res., 230:257, 1988).

The isolation of growth factors and the genes encoding them is importantin the development of diagnostics and therapeutics for variousconnective tissue-related disorders. The present invention provides suchan invention.

SUMMARY OF THE INVENTION

Various cell types produce and secrete PDGF and PDGF-related molecules.In an attempt to identify the type of PDGF dimers present in the growthmedia of cultured endothelial cells, a new growth factor was discovered.This previously unknown factor, termed Connective Tissue Growth Factor(CTGF), is related immunologically and biologically to PDGF, however itis the product of a distinct gene.

In a first aspect, the present invention provides a polypeptide growthfactor for connective tissue cells. The polypeptide is a mitogenic agentand a chemotactic agent for cells.

In a second aspect, the present invention provides a polynucleotideencoding a connective tissue growth factor characterized as encoding aprotein (1) existing as a monomer of approximately 36-38 kD molecularweight, and (2) capable of binding to a PDGF receptor.

In a further aspect, the invention provides a method for acceleratingwound healing in a subject by applying to the wound an effective amountof a composition which contains CTGF.

In yet another aspect, the invention provides a method of diagnosingpathological states in a subject suspected of having pathologycharacterized by a cell proliferative disorder which comprises, (1)obtaining a sample suspected of containing CTGF from the subject, (2)determining the level of CTGF in the sample, and (3) comparing the levelof CTGF in the sample to the level of CTGF in normal tissues.

A method of ameliorating diseases characterized by a cell proliferativedisorder, by treating the site of the disease with an effective amountof a CTGF reactive agent is also provided.

The present invention identifies a TGF-β responsive or regulatoryelement in the 5' untranslated nucleotides of the CTGF gene (about -157to -145). Based on the identification of this element, the invention nowprovides a method for identifying a composition which affects CTGFexpression comprising incubating components comprising the compositionand TGF-β regulatory element (TβRE), in the presence of a TGF-β factorwhich regulates TβRE, and measuring the effect of the composition onCTGF expression. Thus, the invention provides a means for drug discoveryfor treatment of fibrotic diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the structural organization of the CTGF gene. Exons areindicated by boxed regions, with solid areas in the gene correspondingto the open reading frame.

FIGS. 1B through 1E show a comparison of nucleotide sequences betweenCTGF promoter (nucleotides 1 to 840; SEQ ID NO: 1) and fisp-12 promoter(SEQ ID NO: 9). Identical nucleotides are marked with asterisks. TheTATA box and other consensus sequences are indicated and shaded. Thesite of transcriptional initiation is indicated at position number +1.

FIGS. 1C through 1J show the complete nucleotide (SEQ ID NO: 1) anddeduced amino acid (SEQ ID NO: 2) sequence for the CTGF structural geneand 5' and 3' untranslated sequences.

FIGS. 2A-2C shows a Northern blot analysis. Panel (A) shows prolongedinduction of CTGF mRNA by short term TGF-β. Confluent cultures of humanskin fibroblasts were incubated with DMEM-ITS containing 5 μg/ml ofInsulin, 5 μg/ml of Transferrin and 5 ng/ml of Selenium for 24 hoursprior to the addition of TGF-β. After the treatment with 10 ng/ml ofTGF-β for 1 hour, cells were washed with PBS and incubated with DMEM-ITSfor indicated time periods. Panel (B) shows the effect of cycloheximide(CHX) on induction of CTGF mRNA. Lane A and H are non-treated controlcells at 4 hours and 24 hours, respectedly. Lane B, 4 hrs. Cycloheximide(10 ug/ml); Lane C, 4 hrs TGF-β present for 1 hour during hour 1 of 2 ofcycloheximide exposure; Lane E, same as B with RNA prepared 24 hoursafter addition of cycloheximide; Lane f, 24 hours TGF-β (10 ug/ml); LaneG, same as D with RNA prepared 24 hours after addition of cycloheximideand 22 hours after removal of TGF-β. Panel (C) shows the effect ofprotein synthesis inhibitors on induction of CTGF mRNA. Cells weretreated with puromycin or anisomycin for 4 hours. TGF-β was added 1 hourafter the addition of protein synthesis inhibitor and cells wereincubated for 3 hours prior to isolation of total RNA. CTGF transcriptswere analyzed by Northern blot as described in the EXAMPLES.

FIG. 3A shows deletion analysis of CTGF promoter-luciferase constructs.Known consensus sequences are indicated. NIH/3T3 fibroblasts weretransfected with the constructs and 10 ng/ml of TGF-β was added foractivation of the cells and cell extracts were prepared 24 hours later.Relative induction is indicated as fold above non-induced control cellsand normalized using the β-galactosidase activity from control plasmidsthat were cotransfected with the CTGF constructs. These studies wererepeated 6 times with similar results. Data represent the average ofduplicate transfections with the indicated construct performed in asingle set of experiments.

FIG. 3B shows TGF-β response of an SV40 enhancerless promoterelement-luciferase reporter construct containing the TGF-β region of theCTGF promoter. The indicated regions of the CTGF promoter were cloned inboth orientations upstream from an SV40 enhancerless promoter. Cellswere treated with 10 ng/ml of TGF-β for 24 hrs prior to assay forluciferase activity. These experiments were repeated 4 times withsimilar results. Data represents the average of duplicate transfectionsof the indicated construct from a single experimental set.

FIG. 4 shows competitive gel shift assays to delineate TGF-β responseelement in the -205 to -109 region of the CTGF promoter. A nucleotidefragment consisting of the region from -205 to -109 of the CTGF promoterwas end labeled with ³² P and used in competitive gel shifts with theindicated oligonucleotides. The specific gel shifted band is indicatedby the arrow. A diagram of the sequences used indicates the position ofthese corresponding to the NF-1 and TIE like elements. The numberedfragments in the diagram indicate the lane number in the competitive gelshift assay with the specific nucleotide sequence indicated above thelane (i.e. 3, -205/-150). Unlabeled competitors were used at a 250 foldmolar excess over the labeled fragment. Only oligonucleotides containingthe region from -169 to -150 acted as specific competitors. Neither theNF-1 or TIE like regions competed in this assay.

FIG. 5A shows a methylation interference assay of the -205 to -109region of the CTGF promoter. FIG. 5 panel A shows a sequence analysis ofthe region from -205 to -109. Sequence from -200 to -113 is shown. LaneG is the G sequence of the intact labeled probe, Lane S is the sequenceof the shifted band and Lane F is the sequence of the non-shifted freeprobe from the same sample. The only region containing mission Gresidues is from positions -157 to -145.

FIG. 5B shows a sequence analysis of the region -159 to -142 using asmaller fragment of the promoter (-169 to 193). Lanes are the same as inA. Competed G residues in this sequence are indicated by arrows. Solidcircles indicate G residues detected in analysis of complementary strand(data not shown). Symbols * and # are for orientation with sequence inA.

FIG. 6 shows competitive gel shift titration assay of oligonucleotidesin the TβRE. Overlapping and non-overlapping oligonucleotides containingportions of the -159 to -143 region of the CTGF promoter were tested inthe competitive gel shift assay using a ³² P-end labeled human CTGFpromoter fragment (-205 to -109). The intact fragment (-159 to -143)exhibits the highest affinity with complete competition at 10 ng. Allother fragments which contain only a portion of this sequence are lesseffective with the -150 to -134 region being the least effective. Lanes14 and 15 are the NF-1 and TIE like elements respectively and show nocompetition at 5000 fold molar excess of labeled probe.

FIG. 7 shows point mutations in the TβRE decreases induction of the CTGFpromoter by TGF-β.

FIGS. 8A -8D show the effect of herbimycin, phorbol ester, cAMP andcholera toxin on TGF-β induced CTGF expression as measured in aluciferase assay.

FIG. 8E shows photomicrographs of NIH/3T3 cells either untreated,treated with TGF-β, cAMP (8Br cAMP) or cAMP and TGF-β.

FIG. 8F shows the results of inhibition of anchorage independent growthby 8Br cAMP and cholera toxin, and reversal of cAMP or cholera toxininhibition of TGF-β induced anchorage independent growth by CTGF.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses a novel protein growth factor calledConnective Tissue Growth Factor (CTGF). This protein may play asignificant role in the normal development, growth and repair of humantissue. The discovery of the CTGF protein and cloning of the cDNAencoding this molecule is significant in that it is a previously unknowngrowth factor having mitogenic and chemotactic activities for connectivetissue cells. The biological activity of CTGF is similar to that ofPDGF, however, CTGF is the product of a gene unrelated to the A or Bchain genes of PDGF. Since CTGF is produced by endothelial andfibroblastic cells, both of which are present at the site of a wound, itis probable that CTGF functions as a growth factor in wound healing.

Pathologically, CTGF may be involved in diseases in which there is anovergrowth of connective tissue cells, such as cancer, tumor formationand growth, fibrotic diseases (e.g., pulmonary fibrosis, kidneyfibrosis, glaucoma) and atherosclerosis. The CTGF polypeptide is usefulas a therapeutic in cases in which there is impaired healing of skinwounds or there is a need to augment the normal healing mechanisms.Additionally, antibodies to CTGF polypeptide or fragments could bevaluable as diagnostic tools to aid in the detection of diseases inwhich CTGF is a pathological factor. Therapeutically, antibodies orfragments of the antibody molecule could also be used to neutralize thebiological activity of CTGF in diseases where CTGF is inducing theovergrowth of tissue.

The primary biological activity of CTGF polypeptide is its mitogenicity,or ability to stimulate target cells to proliferate. The ultimate resultof this mitogenic activity in vivo, is the growth of targeted tissue.CTGF also possesses chemotactic activity, which is the chemicallyinduced movement of cells as a result of interaction with particularmolecules. Preferably, the CTGF of this invention is mitogenic andchemotactic for connective tissue cells, however, other cell types maybe responsive to CTGF polypeptide as well.

The CTGF polypeptide of the invention is characterized by existing as amonomer of approximately 36-38 kD molecular weight. CTGF is secreted bycells and is active upon interaction with a receptor on a responsivecell. CTGF is antigenically related to PDGF although there is little ifany peptide sequence homology. Anti-PDGF antibody has high affinity tothe non-reduced forms of the PDGF isomers and the CTGF molecule andten-fold less affinity to the reduced forms of these peptides, whichlack biological activity. This suggests that there are regions of sharedtertiary structure between the PDGF isomers and the CTGF molecule,resulting in common antigenic epitopes.

The term "substantially pure" as used herein refers to CTGF which issubstantially free of other proteins, lipids, carbohydrates or othermaterials with which it is naturally associated. The substantially purepolypeptide will yield a single major band on a non-reducingpolyacrylamide gel. The purity of the CTGF polypeptide can also bedetermined by amino-terminal amino acid sequence analysis. CTGFpolypeptide includes functional fragments of the polypeptide, so long asthe mitogenic and chemotactic activities of CTGF are retained. Smallerpeptides containing the biological activity of CTGF are included in theinvention. Additionally, more effective CTGF molecules produced, forexample, through site directed mutagenesis of the CTGF cDNA areincluded.

The invention provides an isolated polynucleotide encoding the CTGFprotein. The term "isolated" as used herein refers to a polynucleotidewhich is substantially free of other polynucleotides, proteins, lipids,carbohydrates or other materials with which it is naturally associated.These polynucleotides include DNA, cDNA and RNA sequences which encodeconnective tissue growth factor. It is understood that allpolynucleotides encoding all or a portion of CTGF are also includedherein, so long as they encode a polypeptide with the mitogenic andchemotactic activity of CTGF. Such polynucleotides include naturallyoccurring forms, such as allelic variants, and intentionally manipulatedforms, for example, mutagenized polynucleotides, as well as artificiallysynthesized polynucleotides. For example, CTGF polynucleotide may besubjected to site-directed mutagenesis. The polynucleotides of theinvention include sequences that are degenerate as a result of thegenetic code. There are only 20 natural amino acids, most of which arespecified by more than one codon. Therefore as long as the amino acidsequence of CTGF is functionally unchanged, all degenerate nucleotidesequences are included in the invention.

The term "polynucleotide" also denotes DNA, cDNA and RNA which encodeuntranslated sequences which flank the structural gene encoding CTGF.For example, a polynucleotide of the invention includes 5' regulatorynucleotide sequences and 3' untranslated sequences associated with theCTGF structural gene. The polynucleotide of the invention which includesthe 5' and 3' untranslated region is illustrated in FIG. 1C. The 5'regulatory region, including the promoter, is illustrated in FIG. 1B.

The sequence of the cDNA for CTGF contains an open reading frame of 1047nucleotides with an initiation site at position 130 and a TGAtermination site at position 1177 and encodes a peptide of 349 aminoacids. There is only a 40% sequence homology between the CTGF cDNA andthe cDNA for both the A and B chains of PDGF.

The present invention provides CTGF promoter nucleotides -823 to +74(nucleotides 1 to 897; SEQ ID NO: 1) as well as a TGF-β regulatoryelement (TβRE) located between positions -162 and -128 (nucleotides 664to 698; SEQ ID NO: 1) of the CTGF promoter sequence. Methylationinterference and competition gel shift assays map a unique 13-nucleotidesequence between positions -157 and -145 (nucleotides 669 to 681; SEQ IDNO. 1) delineating a novel TGF-β cis-regulatory element.

The CTGF open reading frame encodes a polypeptide which contains 39cysteine residues, indicating a protein with multiple intramoleculardisulfide bonds. The amino terminus of the peptide contains ahydrophobic signal sequence indicative of a secreted protein and thereare two N-linked glycosylation sites at asparagine residues 28 and 225in the amino acid sequence. CTGF is a member of a protein family thatincludes serum induced immediate early gene products such as Cyr61(O'Brien, et al., Mol. Cell. Biol., 10:3569, 1990) and Fisp12 (Ryseck,et al., Cell Growth & Differentiation, 2:225, 1991)/BigM2 (Brunner, etal., DNA and Cell Biol., 10:293, 1991); a v-src induced peptide (CEF-10)(Simmons, et al., Proc. Natl. Acad. Sci., USA, 86:1178, 1989) and aputative oncoprotein (nov) (Joliot, et al., Mol. Cell Biol, 12:10,1992). Twisted gastrulation (tsg), a gene that functions to control theinduction of medial mesodermal elements in the dorsal/ventral patterningof Drosophila embryogenesis is more distantly related to CTGF (Mason, etal., Genes and Devel, 8:1489, 1994). There is a 45% overall sequencehomology between the CTGF polypeptide and the polypeptide encoded by theCEF-10 mRNA transcript (Simmons, et al., Proc. Natl. Acad. Sci. USA86:1178, 1989); the homology reaches 52% when a putative alternativesplicing region is deleted.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization procedures whichare well known in the art. These include, but are not limited to: 1)hybridization of probes to genomic or cDNA libraries to detect sharednucleotide sequences and 2) antibody screening of expression librariesto detect shared structural features.

Screening procedures which rely on nucleic acid hybridization make itpossible to isolate any gene sequence from any organism, provided theappropriate probe is available. For example, oligonucleotide probes,which correspond to a part of the sequence encoding the protein inquestion, can be synthesized chemically. This requires that short,oligopeptide, stretches of amino acid sequence must be known. The DNAsequence encoding the protein can be deduced from the genetic code,however, the degeneracy of the code must be taken into account. It ispossible to perform a mixed addition reaction when the sequence isdegenerate. This includes a heterogeneous mixture of denatureddouble-stranded DNA. For such screening, hybridization is preferablyperformed on either single-stranded DNA or denatured double-strandedDNA. Hybridization is particularly useful in the detection of cDNAclones derived from sources where an extremely low amount of mRNAsequences relating to the polypeptide of interest are present. In otherwords, by using stringent hybridization conditions directed to avoidnon-specific binding, it is possible, for example, to allow theautoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucleic AcidResearch, 9:879, 1981).

A cDNA expression library, such as lambda gt11, can be screenedindirectly for CTGF peptides having at least one epitope, usingantibodies specific for CTGF or antibodies to PDGF which cross reactwith CTGF. Such antibodies can be either polyclonally or monoclonallyderived and used to detect expression product indicative of the presenceof CTGF cDNA.

DNA sequences encoding CTGF can be expressed in vitro by DNA transferinto a suitable host cell. "Host cells" are cells in which a vector canbe propagated and its DNA expressed. The term also includes any progenyof the subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term"host cell" is used.

DNA sequences encoding CTGF can be expressed in vivo in eitherprokaryotes or eukaryotes. Methods of expressing DNA sequences havingeukaryotic coding sequences in prokaryotes are well known in the art.Hosts include microbial, yeast and mammalian organisms.

Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectorsare used to incorporate DNA sequences of the invention. In general,expression vectors containing promotor sequences which facilitate theefficient transcription of the inserted eukaryotic genetic sequence areused in connection with the host. The expression vector typicallycontains an origin of replication, a promoter, and a terminator, as wellas specific genes which are capable of providing phenotypic selection ofthe transformed cells.

In addition to expression vectors known in the art such as bacterial,yeast and mammalian expression systems, baculovirus vectors may also beused. One advantage to expression of foreign genes in this invertebratevirus expression vector is that it is capable of expression of highlevels of recombinant proteins, which are antigenically and functionallysimilar to their natural counterparts. Baculovirus vectors and theappropriate insect host cells used in conjunction with the vectors willbe known to those skilled in the art.

The term "recombinant expression vector" refers to a plasmid, virus orother vehicle known in the art that has been manipulated by insertion orincorporation of the CTGF genetic sequences. Such expression vectorscontain a promoter sequence which facilitates the efficienttranscription of the inserted genetic sequence of the host. Theexpression vector typically contains an origin of replication, apromoter, as well as specific genes which allow phenotypic selection ofthe transformed cells. Vectors suitable for use in the present inventioninclude, but are not limited to the T7-based expression vector forexpression in bacteria (Rosenberg, et al., Gene, 56:125, 1987), thepMSXND expression vector for expression in mammalian cells (Lee andNathans, J. Biol. Chem., 263:3521, 1988) and baculovirus-derived vectorsfor expression in insect cells. The DNA segment can be present in thevector operably linked to regulatory elements, for example, a promoter(e.g., T7, metallothionein I, or polyhedrin promoters).

The vector may include a phenotypically selectable marker to identifyhost cells which contain the expression vector. Examples of markerstypically used in prokaryotic expression vectors include antibioticresistance genes for ampicillin (β-lactamases), tetracycline andchloramphenicol (chloramphenicol acetyltransferase). Examples of suchmarkers typically used in mammalian expression vectors include the genefor adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo,G418), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase(HPH), thymidine kinase (TK), and xanthine guaninephosphoribosyltransferse (XGPRT, gpt).

The isolation and purification of host cell expressed polypeptides ofthe invention may be by any conventional means such as, for example,preparative chromatographic separations and immunological separationssuch as those involving the use of monoclonal or polyclonal antibody.

Transformation of the host cell with the recombinant DNA may be carriedout by conventional techniques well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth and subsequently treated by the CaCl₂ method usingprocedures well known in the art. Alternatively, MgCl₂ or RbCl could beused.

Where the host used is a eukaryote, various methods of DNA transfer canbe used. These include transfection of DNA by calciumphosphate-precipitates, conventional mechanical procedures such asmicroinjection, insertion of a plasmid encased in liposomes, or the useof virus vectors. Eukaryotic cells can also be cotransformed with DNAsequences encoding the polypeptides of the invention, and a secondforeign DNA molecule encoding a selectable phenotype, such as the herpessimplex thymidine kinase gene. Another method is to use a eukaryoticviral vector, such as simian virus 40 (SV40) or bovine papilloma virus,to transiently infect or transform eukaryotic cells and express theprotein. (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory,Gluzman ed., 1982). Examples of mammalian host cells include COS, BHK,293, and CHO cells.

Eukaryotic host cells may also include yeast. For example, DNA can beexpressed in yeast by inserting the DNA into appropriate expressionvectors and introducing the product into the host cells. Various shuttlevectors for the expression of foreign genes in yeast have been reported(Heinemann, J. et al., Nature, 340:205, 1989; Rose, M. et al., Gene,60:237, 1987).

The invention provides antibodies which are specifically reactive withCTGF polypeptide or fragments thereof. Although this polypeptide iscross reactive with antibodies to PDGF, not all antibodies to CTGF willalso be reactive with PDGF. Antibody which consists essentially ofpooled monoclonal antibodies with different epitopic specificities, aswell as distinct monoclonal antibody preparations are provided.Monoclonal antibodies are made from antigen containing fragments of theprotein by methods well known in the art (Kohler, et al., Nature,256:495, 1975; Current Protocols in Molecular Biology, Ausubel, et al.,ed., 1989). Monoclonal antibodies specific for CTGF can be selected, forexample, by screening for hybridoma culture supernatants which reactwith CTGF, but do not react with PDGF.

Antibody which consists essentially of pooled monoclonal antibodies withdifferent epitopic specificities, as well as distinct monoclonalantibody preparations are provided. Monoclonal antibodies are made fromantigen containing fragments of the protein by methods well known in theart (Kohler, et al., Nature, 256:495, 1975; Current Protocols inMolecular Biology, Ausubel, et al., ed., 1989).

The term "antibody" as used in this invention includes intact moleculesas well as fragments thereof, such as Fab, F(ab')₂, and Fv which arecapable of binding the epitopic determinant. These antibody fragmentsretain some ability to selectively bind with its antigen or receptor andare defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab', the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab' fragmentsare obtained per antibody molecule;

(3) (Fab')₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab')₂ is a dimer of two Fab' fragments held together by twodisulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; and

(5) Single chain antibody ("SCA"), defined as a genetically engineeredmolecule containing the variable region of the light chain, the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), incorporated herein by reference).

As used in this invention, the term "epitope" means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

Antibodies which bind to CTGF polypeptide of the invention can beprepared using an intact polypeptide or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide or apeptide used to immunize an animal can be derived from translated cDNAor chemical synthesis which can be conjugated to a carrier protein, ifdesired. Such commonly used carriers which are chemically coupled to thepeptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovineserum albumin (BSA), and tetanus toxoid. The coupled peptide is thenused to immunize the animal (e.g., a mouse, a rat, or a rabbit).

If desired, polyclonal or monoclonal antibodies can be further purified,for example, by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those of skill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies (See for example, Coligan,et al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1994, incorporated herein by reference).

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the"image" of the epitope bound by the first monoclonal antibody.

The invention provides a method for accelerating wound healing in asubject, e.g., a human, by applying to the wound an effective amount ofa composition which contains CTGF, preferably purified. PDGF andPDGF-related molecules, such as CTGF, are involved in normal healing ofskin wounds. The CTGF polypeptide of this invention is valuable as atherapeutic in cases in which there is impaired healing of skin woundsor there is a need to augment the normal healing mechanisms, e.g.,burns. One important advantage to using CTGF protein to accelerate woundhealing is attributable to the molecule's high percentage of cysteineresidues. CTGF, or functional fragments thereof, is more stable and lesssusceptible to protease degradation than PDGF and other growth factorsknown to be involved in wound healing.

CTGF is produced by endothelial cells and fibroblastic cells, both ofwhich are present at the site of a skin wound. Therefore, agents whichstimulate the production of CTGF can be added to a composition which isused to accelerate wound healing. Preferably, the agent of thisinvention is transforming growth factor beta (TGF-β), however, it islikely that other TGF-β family members will also be useful inaccelerating wound healing by inducing CTGF. The composition of theinvention aids in healing the wound, in part, by promoting the growth ofconnective tissue. The composition is prepared by combining, in apharmaceutically acceptable carrier substance, e.g., inert gels orliquids, the purified CTGF and TGF-β.

The term "cell proliferative disorder" refers to pathological statescharacterized by the continual multiplication of cells resulting in anovergrowth of a cell population within a tissue. The cell populationsare not necessarily transformed, tumorigenic or malignant cells, but caninclude normal cells as well. For example, CTGF may be involvedpathologically by inducing a proliferative lesion in the intimal layerof an arterial wall, resulting in atherosclerosis. Instead of trying toreduce risk factors for the disease, e.g., lowering blood pressure orreducing elevated cholesterol levels in a subject, CTGF inhibitors orantagonists of the invention would be useful in interfering with the invivo activity of CTGF associated with atherosclerosis. CTGF antagonistsare useful in treating other disorders associated with overgrowth ofconnective tissues, such as various fibrotic diseases, includingscleroderna, arthritis, alcoholic liver cirrhosis, keloid, andhypertropic scar.

The present invention provides a method to detect the presence ofelevated levels of CTGF to be used diagnostically to determine thepresence of pathologies characterized by a cell proliferative disorder.For example, a sample suspected of containing CTGF is obtained from asubject, the level of CTGF determined and this level is compared withthe level of CTGF in normal tissue. The level of CTGF can be determinedby immunoassays using anti-CTGF antibodies, for example. Othervariations of such assays which are well known to those skilled in theart, such as radioimmunoassay (RIA), ELISA and immunofluorescence canalso be used to determine CTGF levels in a sample. Alternatively,nucleic acid probes can be used to detect and quantitate CTGF mRNA forthe same purpose.

The invention also discloses a method for ameliorating diseasescharacterized by a cell proliferative disorder by treating the site ofthe disease with an effective amount of a CTGF reactive agent. The term"ameliorate" denotes a lessening of the detrimental effect of thedisease-inducing response in the patient receiving therapy. Where thedisease is due to an overgrowth of cells, an antagonist of CTGFpolypeptide is effective in decreasing the amount of growth factor thatcan bind to a CTGF specific receptor on a cell. Such an antagonist maybe a CTGF specific antibody or functional fragments thereof (e.g., Fab,F(ab')₂). Alternatively, a polynucleotide containing the TβRE region ofthe promoter may be used as a CTGF reactive agent by acting as acompetitor for TGF-β. The treatment requires contacting the site of thedisease with the antagonist. Where the cell proliferative disorder isdue to a diminished amount of growth of cells, a CTGF reactive agentwhich is stimulatory is contacted with the site of the disease. Forexample, TGF-β is one such reactive agent. Other agents will be known tothose skilled in the art.

When a cell proliferative disorder is associated with the expression ofCTGF, a therapeutic approach which directly interferes with thetranslation of CTGF messages into protein is possible. For example,antisense nucleic acid or ribozymes could be used to bind to the CTGFmRNA or to cleave it. Antisense RNA or DNA molecules bind specificallywith a targeted gene's RNA message, interrupting the expression of thatgene's protein product. The antisense binds to the messenger RNA forminga double stranded molecule which cannot be translated by the cell.Antisense oligonucleotides of about 15-25 nucleotides are preferredsince they are easily synthesized and have an inhibitory effect justlike antisense RNA molecules. In addition, chemically reactive groups,such as iron-linked ethylenediaminetetraacetic acid (EDTA-Fe) can beattached to an antisense oligonucleotide, causing cleavage of the RNA atthe site of hybridization. These and other uses of antisense methods toinhibit the in vitro translation of genes are well known in the art(Marcus-Sakura, Anal., Biochem., 172:289, 1988).

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub, ScientificAmerican, 262:40, 1990). In the cell, the antisense nucleic acidshybridize to the corresponding mRNA, forming a double-stranded molecule.The antisense nucleic acids interfere with the translation of the mRNA,since the cell will not translate a mRNA that is double-stranded.Antisense oligomers of about 15 nucleotides are preferred, since theyare easily synthesized and are less likely to cause problems than largermolecules when introduced into the target CTGF producing cell. The useof antisense methods to inhibit the in vitro translation of genes iswell known in the art (Marcus-Sakura, Anal.Biochem., 172:289, 1988).

Use of an oligonucleotide to stall transcription is known as the triplexstrategy since the oligomer winds around double-helical DNA, forming athree-strand helix. Therefore, these triplex compounds can be designedto recognize a unique site on a chosen gene (Maher, et al., AntisenseRes. and Dev., 1(3):227, 1991; Helene, C., Anticancer Drug Design,6(6):569, 1991) for example, the TβRE region of the CTGF promoter.

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J.Amer.Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and "hammerhead"-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while "hammerhead"-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

The identification of the promoter element of the CTGF gene andspecifically, the TGF-β responsive/regulatory element (TβRE)(5'-GTGTCAAGGGGTC-3' (SEQ ID NO:8); nucleotides -157 and -145), providesa source for a screening method for identifying compounds orcompositions which affect the expression of CTGF. Thus, in anotherembodiment, the invention provides a method for identifying acomposition which affects CTGF expression comprising incubatingcomponents comprising the composition and a TGF-β responsive element ofthe CTGF promoter, wherein the incubating is carried out underconditions sufficient to allow the components to interact; and measuringthe effect of the composition on CTGF expression. The method furthercomprises adding TGF-β, or a TGF-β family member reactive with the TβRE,to the reaction mixture. Therefore, the method allows identification ofTGF-β inhibitors, or anti-fibrotic compounds. Preferably, the promoterregion used in the screening assays described herein includesnucleotides -823 to +74, however, smaller regions that include the TGF-βresponsive element would also be useful in the method of the invention(e.g., -162 to -128 or -157 to -145).

The observed effect on CTGF expression may be either inhibitory orstimulatory. For example, the increase or decrease of CTGF activity canbe measured by a biological assay for CTGF, as described in the examplesherein (e.g., EXAMPLES 1 and 2). Alternatively, a polynucleotideencoding both the regulatory (promoter) and structural region of CTGFmay be inserted into an expression vector and the effect of acomposition on transcription of CTGF can be measured, for example, byNorthern blot analysis. A radioactive compound is added to the mixtureof components, such as ³² P-ATP, and radioactive incorporation into CTGFmRNA is measured.

Alternatively, a composition which affects the expression of CTGF can beidentified by operably linking a reporter gene with the TGF-β responsiveregion of the promoter of CTGF, incubating the components including thecomposition being tested, the reporter gene construct and TGF-β andassaying for expression of the reporter gene. Such reporter genes willbe known to those of skill in the art, and include but are not limitedto a luciferase gene, chloramphenicol acetyl transferase gene (CATassay) or β-galactosidase gene.

The inducer of the TβRE can be added prior to or following the additionof the composition to be tested. Preferably, it is added after thecomposition is added. An inducer of this region in the CTGF promoter ispreferably TGF-β, however, it is likely that other members of the TGF-βfamily will also be useful for induction from this element. Other suchfamily members or factors will be known to those of skill in the art.

The method of the invention is preferably performed in an indicatorcell. An "indicator cell" is one in which activation of CTGF or thereporter gene can be detected. Examples of mammalian host indicatorcells include the pre-B cell line, 70Z/3, Jurkat T, COS, BHK, 293, CHO,HepG2, and HeLa cells. Other cell lines can be utilized as indicatorcells, as long as the level of reporter gene can be detected. The cellscan be recombinantly modified to contain an expression vector whichencodes one or more additional copies of the TβRE binding motif,preferably operatively linked to a reporter gene. The cells can also bemodified to express CTGF, as described above.

The reporter gene is a phenotypically identifiable marker for detectionof stimulation or inhibition of CTGF activation. Markers preferably usedin the present invention include the LUC gene whose expression isdetectable by a luciferase assay. Examples of markers typically used inprokaryotic expression vectors include antibiotic resistance genes forampicillin (β-lactamases), tetracycline and chloramphenicol(chloramphenicol acetyl-transferase). Examples of such markers typicallyused in mammalian expression vectors, which are preferable for thepresent invention, include the gene for adenosine deaminase (ADA),aminoglycoside phosphotransferase (neo, G418), dihydrofolate reductase(DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK),xanthine guanine phosphoribosyltransferse (XGPRT, gpt) andβ-galactosidase (β-gal).

In yet another embodiment, the invention provides a method of treating asubject having a cell proliferative disorder associated with CTGF geneexpression in a subject, comprising administering to a subject havingthe disorder a therapeutically effective amount of an agent whichmodulates CTGF gene expression, thereby treating the disorder. The term"modulate" refers to inhibition or suppression of CTGF expression whenCTGF is overexpressed, and induction of expression when CTGF isunderexpressed. The term "therapeutically effective" means that amountof CTGF agent which is effective in reducing the symptoms of the CTGFassociated cell proliferative disorder.

The agent which modulates CTGF gene expression may be a polynucleotidefor example. The polynucleotide may be an antisense, a triplex agent, ora ribozyme, as described above. For example, an antisense may bedirected to the structural gene region or to the promoter region ofCTGF.

The agent also includes a polynucleotide which includes the TβRE of theinvention. Preferably this region corresponds to nucleotides -162 to-128 of the CTGF regulatory polypeptide illustrated in FIG. 1B. Morespecifically, the TβRE region corresponds to about -157 to -145 in FIG.1B. These polynucleotides are useful as competitive inhibitors orpseudosubstrates for TGF-β or other growth factors which bind to theTβRE and induce CTGF transcription.

Delivery of antisense, triplex agents, ribozymes, achieved using a recomand the like can be achieved using a recombinant expression vector suchas a chimeric virus or a colloidal dispersion system. Various viralvectors which can be utilized for gene therapy as taught herein includeadenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such asa retrovirus. Preferably, the retroviral vector is a derivative of amurine or avian retrovirus. Examples of retroviral vectors in which asingle foreign gene can be inserted include, but are not limited to:Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus(RSV). A number of additional retroviral vectors can incorporatemultiple genes. All of these vectors can transfer or incorporate a genefor a selectable marker so that transduced cells can be identified andgenerated. By inserting a polynucleotide sequence of interest into theviral vector, along with another gene which encodes the ligand for areceptor on a specific target cell, for example, the vector is nowtarget specific. Retroviral vectors can be made target specific byinserting, for example, a polynucleotide encoding a sugar, a glycolipid,or a protein. Preferred targeting is accomplished by using an antibodyto target the retroviral vector. Those of skill in the art will know of,or can readily ascertain without undue experimentation, specificpolynucleotide sequences which can be inserted into the retroviralgenome to allow target specific delivery of the retroviral vectorcontaining the antisense polynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsidation. Helper cell lines which havedeletions of the packaging signal include but are not limited to Ψ2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced.

Alternatively, NIH 3T3 or other tissue culture cells can be directlytransfected with plasmids encoding the retroviral structural genes gag,pol and env, by conventional calcium phosphate transfection. These cellsare then transfected with the vector plasmid containing the genes ofinterest. The resulting cells release the retroviral vector into theculture medium.

Another targeted delivery system for antisense polynucleotides acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. It has beenshown that large unilamellar vesicles (LUV), which range in size from0.2-4.0 um can encapsulate a substantial percentage of an aqueous buffercontaining large macromolecules. RNA, DNA and intact virions can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form (Fraley, et al., Trends Biochem. Sci., 6:77,1981). In addition to mammalian cells, liposomes have been used fordelivery of polynucleotides in plant, yeast and bacterial cells. Inorder for a liposome to be an efficient gene transfer vehicle, thefollowing characteristics should be present: (1) encapsulation of thegenes of interest at high efficiency while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes has been classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand. In general, the compounds bound tothe surface of the targeted delivery system will be ligands andreceptors which will allow the targeted delivery system to find and"home in" on the desired cells. A ligand may be any compound of interestwhich will bind to another compound, such as a receptor.

The agent which modulates CTGF gene expression in the method of theinvention includes agents which cause an elevation in cyclic nuclotidesin the cell. For example, agents such as cholera toxin or 8Br-cAMP arepreferably administered to a subject having a cell proliferativedisorder associated with CTGF gene expression. Preferably, the cyclicnucleotide that is elevated after treatment in the method of theinvention is cAMP or a cAMP analog, either functional or structural, orboth. Those of skill in the art will know of other agents which inducecAMP or similar analogs in a cell and which are useful in the method ofthe invention.

The therapeutic agents useful in the method of the invention can beadministered parenterally by injection or by gradual perfusion overtime. Administration may be intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, antimicrobials, anti-oxidants, chelating agents and inert gasesand the like.

The invention also includes a pharmaceutical composition comprising atherapeutically effective amount of CTGF in a pharmaceuticallyacceptable carrier. Such carriers include those listed above withreference to parenteral administration.

The present Examples (see EXAMPLE 10), demonstrate that TGF-β inductionof CTGF is cell type specific (e.g., fibroblast). Consequently, the CTGFpromoter region, including the TβRE, is useful for the expression of astructural gene specifically in connective tissue cells. It isenvisioned that any gene product of interest can be specificallyproduced in a connective tissue cell, once operably linked to the TβRE,and in the presence of TGF-β. For example, it may be desirable tooperably link PDGF or another growth factor to a polynucleotidecontaining TβRE, thereby specifically producing PDGF or another factorin a connective tissue cell. Alternatively, in cases where the level ofCTGF or other factor produced is elevated, it may be desirable tointroduce an antisense for CTGF, for example, under control of TβRE,thereby decreasing the production of CTGF in the cell.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLE 1 Identification and Partial Purification of Mitogen from HUVECells PDGF-Immunorelated

Cells

Human umbilical vein endothelial (HUVE) cells were isolated from freshhuman umbilical cords by collagenase perfusion (Jaffe, et al., HumanPathol., 18:234, 1987) and maintained in medium 199 with 20% FCS, 0.68mM L-glutamine, 20 μg/ml Gentamicin, 90 μg/ml porcine heparin (Sigma,St. Louis, Mo.), and 50 μg/ml Endothelial Cell Growth Supplement(Sigma). Cells used for media collection were third passage cells. Cellswere identified as endothelial cells by their non-overlappingcobblestone morphology and by positive staining for Factor-VIII relatedantigen. NRK cells were obtained from American Type Culture, NIH/3T3cells were a gift from S. Aaronson (NCI; Bethesda, Md.), and both celllines were maintained in DMEM, 10% FCS, 20 μg/ml Gentamicin. Fetalbovine aortic smooth muscle cells were obtained from tissue explants aspreviously described (Grotendorst, et al., Proc. Natl. Acad. Sci. USA,78 :3669, 1981) and maintained in DMEM, 10% FCS, 20 μg/ml Gentamicin,and used in assays at second or third passage.

Growth Factors and Antibodies

Human PDGF was purified to homogeneity from platelets as describedpreviously (Grotendorst, Cell, 36:279, 1984). Recombinant AA, BB, and ABchain dimeric PDGF molecules were obtained from Creative Biomolecules,(Hopkinton, Mass.). FGF was obtained from Sigma. Purified PDGF orsynthetic peptides containing the amino and carboxyl sequences of themature PDGF A and B chain molecules were used to raise antibodies ingoats. Goats were immunized with 20 μg of purified PDGF or 50 μg ofsynthetic peptide in Freunds complete adjuvant by multiple intradermalinjections. Immune sera were collected seven days after the fourthrechallenge (in Freunds incomplete adjuvant) and subsequentrechallenges. The anti-PDGF antibody did not show any cross-reactivityto TGF-β, EGF, or FGF in immunoblot analysis. The anti-peptideantibodies were sequence specific and did not cross-react with othersynthetic peptide sequences or with recombinant PDGF peptides which didnot contain the specific antigenic sequence. This was determined byWestern blot and dot blot analysis.

Antibody Affinity Column

Goat anti-human PDGF IgG (150 mg) was covalently bound to 25 mls ofAffi-Gel 10 support (BioRad) according to the manufacturers instructionswith a final concentration of 6 mg IgG/ml gel. The column was incubatedwith agitation at 4° C. for 18 hours with 1 liter of HUVE cell mediawhich had been conditioned for 48 hours. The gel was then poured into acolumn (5×1.5 cm), washed with four volumes of 0.1 N acetic acid made pH7.5 with ammonium acetate, and the antibody-bound PDGF immunoreactiveproteins eluted with 1 N acetic acid. Peak fractions were determined bybiological assays and immunoblotting and the fractions pooled.

Initial studies of the PDGF-related growth factors secreted by HUVEcells were done by removing the serum containing growth media fromconfluent cultures of cells and replacing it with serum-free media.Aliquots of this media were removed periodically and the proteinsimmunoblotted using an antibody specific for human platelet PDGF. Thisantibody does not cross-react with any other known growth factors and isable to detect less than 500 picograms of dimeric PDGF or 10 nanogramsof reduced, monomeric A or B chain peptide on immunoblots. HUVE cellswere grown to confluence in 6 well plates. The growth media was removed,cells washed with PBS and 1 ml of serum-free media was added to eachwell. The media was removed after conditioning for the period of timefrom 6-48 hours, dialyzed against 1 N acetic acid and lyophilized. Thesamples were then run on 12% PAGE, electroblotted to nitrocellulose andvisualized with the anti-human PDGF antibody. Five nanograms of purifiedplatelet PDGF was run as reference.

The results indicated constitutive secretion of several species ofmolecules which are inmmunologically similar to platelet PDGF but are ofhigher relative molecular weight (36-39 kD) than the expected 30-32 kDMW of platelet PDGF or A chain or B chain homodimers. Chemotactic andmitogenic assays performed with this serum-free conditioned mediaindicated the total biological activity present was equivalent to 15ng/ml of platelet PDGF after a 48 hour conditioning period. Incubationof the media with 30 μg/ml of anti-human PDGF IgG neutralizedapproximately 20-30% of the mitogenic activity and similar amount of thechemotactic activity.

The presence in HUVE culture media of several species of PDGFimmunoreactive molecules was unexpected, particularly molecules ofhigher molecular weight than those of the A and B chain dimericmolecules anticipated to be produced and secreted by endothelial cells(Collins, et al., Nature, 328:621-624, 1987; Sitaras, et al., J. Cell.Physiol., 132:376-380, 1987). In order to obtain greater amounts of thePDGF-like proteins for further analysis, the HUVE cells had to be keptin media containing 20% fetal calf serum, since the cells begin to dieafter 24 hours in serum-free or low serum media. The PDGF immunoreactiveproteins were partially purified from the serum containing media by useof an antibody affinity column made with the anti-human PDGF IgG and anAffi-Gel 10 support (BioRad). Mitogenic assays were performed using NRKcells as target cells (PDGF BB=5 ng/ml, PDGF AA=10 ng/ml). HUVE mediawas 250 μl of HUVE cell serum-free conditioned media (48 hours) whichwas dialyzed against 1 N acetic acid, lyophilized, and resuspended inDMEM before addition to test wells. Affinity purified fraction was 5μl/ml of combined, concentrated major pool from Affi-Gel 10 affinitycolumn. Anti-PDGF IgG or non-immune IgG (30 μg/ml) was added to thesamples and incubated 18 hours at 4° C. prior to testing in themitogenic assay. The mean of triplicate samples was determined and thestandard deviation was less than 5%. The experiments were repeated atleast three times with similar results.

When aliquots of the partially purified proteins were assayed forchemotactic and mitogenic activity, all biological activity could beneutralized by prior incubation of the proteins with the anti-human PDGFantibody. This indicated that the only biologically active moleculespresent in the partially purified media proteins were PDGF immunorelatedmolecules.

Aliquots of the partially purified proteins were immunoblotted using thesame anti-PDGF antibody and the data indicated the presence of thehigher MW molecules observed in the serum-free conditioned media. Themajor species secreted migrates on polyacrylamide gels at 36 kD andcomprises at least 50% of the total immunoreactive protein purified fromconditioned media. The immunoreactive species migrating at 37 and 39 kDconstitute most of the remaining immunoreactive protein. A similarpattern was seen with proteins labeled with ³⁵ S-cysteine and affinitypurified with the anti-PDGF IgG immunoaffinity column. Less than 15% ofthe total affinity purified proteins co-migrated with purified plateletPDGF or recombinant PDGF isoforms.

Prior incubation of the antibody with purified PDGF (300 ng PDGF/2 μgIgG) blocked antibody binding to all of the molecules, indicating sharedantigenic determinants with dimeric platelet PDGF. Interestingly, whenthe antibody was blocked with recombinant AA, BB, or AB dimers, antibodybinding to the HUVE secreted proteins was inhibited equally by all threedimeric forms, suggesting that the antibody recognizes common epitopespresent on all three PDGF dimers and the HUVE secreted molecules. Inorder to insure that none of the antibody binding molecules detected onWestern blots was derived from fetal calf serum or other additives inthe culture media, a new, unused antibody affinity column was made andmedia which was never conditioned by cells was processed exactly as theconditioned media. No PDGF immunoreactive molecules were detected in thefractions from this column by immunoblot and no biological activity wasdetected. When platelet PDGF or the recombinant dimers are reduced with200 mM dithiothreitol (DTT), monomeric A chain (17 kD) and B chain (14kD) peptides are observed on immunoblots. Treating the HUVE molecules ina 100 mM DTT sample buffer resulted in slower migration of the majorimmunoreactive peptides on polyacrylamide gels. Most of theimmunoreactive molecules migrated at 38-39 kD and less intense bandswere observed at 25 and 14 kD. It was necessary to run at least 10 timesas much reduced protein as nonreduced in order to detect the reducedmolecules. This is consistent with the affinity of the antibody formonomeric forms of the PDGF A and B chain peptides. These data indicatethat the major species in the PDGF-related affinity purified proteinsfrom conditioned media of HUVE cells was monomeric peptide whichmigrates on acrylamide gels at an apparent molecular weight of 36 kDnonreduced and 38 kD when reduced.

EXAMPLE 2 Biological Assays

Chemotactic activity was determined in the Boyden chamber chemotaxisassay with NIH 3T3 or bovine aortic smooth muscle (BASM) cells asdescribed (Grotendorst, et al., Proc. Natl. Acad. Sci. USA,78:3669-3672, 1981; Grotendorst, et al., Methods in Enzymol.,147:144-152, 1987). Mitogenic assays were performed using 96 well platesand normal rat kidney (NRK) fibroblasts or NIH 3T3 cells as targetcells. The cells were plated in DMEM, 10% FCS; NRK cell cultures wereused 10-14 days after confluence and 3T3 cells made quiescent byincubation for 2 days in serum-free DMEM, 0.2 mg/ml BSA before use.Sample proteins and dilutions of known standards were added to the wellsand the plates incubated at 37° C. in 10% CO₂, 90% air for 18 hours,after which ³ H-thymidine at a final concentration of 5 uCi/ml was addedand incubated for an additional 2 hours. The media was removed, thecells washed and DNA synthesis determined from the ³ H-thymidineincorporation into trichloroacetic acid precipitable material byscintillation counting.

Gel Electrophoresis and Immunoblotting

Electrophoresis was performed on 12% polyacrylamide gels containing SDS(Laemmli, U.K., Nature, 227:680-685, 1970) unless otherwise stated.Immunoblotting was performed by electroblotting the proteins to anitrocellulose membrane and incubating the membrane in 50 mM Tris-HCl,pH 7.4, 100 mM NaCl (TBS) with 5% non-fat dry milk at 25° C. for 1 hourto block non-specific antibody binding. The blocking solution wasremoved and the antibody (15 μg/ml) added in TBS containing 0.5% non-fatdry milk and 1 μg/ml sodium azide and incubated overnight at 25° C. Themembranes were washed 5 times in TBS, 0.5% milk for 10 minutes each washand then incubated with alkaline phosphatase conjugated affinitypurified rabbit anti-goat IgG (KPL, Gaithersburg, Md.) at a 1:1000dilution in TBS containing 0.5% milk at 25° C. for 1 hour. The filterswere washed with TBS five times, 10 minutes each time, and the blotdeveloped using an alkaline phosphatase substrate solution (0.1 MTris-HCl, pH 9, 0.25 mg/ml nitro blue tetrazolium, 0.5 mg/ml 5bromo-4-chloro-3-indolyl phosphate).

Major Chemotactic and Mitogenic Activity Is Produced by 36 kD Peptideand Not PDGF Peptides

In order to determine if the chemotactic and mitogenic activitiesobserved in the partially purified media proteins were from moleculescontaining the PDGF A and B chain peptides or were the products ofmolecules which do not contain these sequences, biological assays wereperformed with serial dilutions of the affinity purified media proteinsand serial dilutions of recombinant PDGF AA and BB homodimers and the ABheterodimer. Sufficient quantities of the samples were prepared toperform the mitogenic and chemotactic assays and the immunoblots withaliquots of each dilution sample. The mitogenic activity of the HUVEaffinity purified factors observed was comparable to the activityelicited by all three recombinant PDGF dimers. The chemotactic activitywas comparable to the AB heterodimer, producing less response than theBB homodimer and greater response than the AA homodimer. When thebiological activity of the samples was compared with immunoblots ofequivalent amounts of the same samples, no A chain nor B chain moleculeswere detected in the test samples. These data demonstrate the majorbiological activity present in the anti-PDGF affinity purified fractioncannot be accounted for by PDGF A or B chain containing molecules andimply that the major PDGF-immunoreactive protein species present inthese samples (the 36 kD peptide) is biologically active and does notcontain amino acid sequences found in the amino and carboxy terminals ofthe PDGF A or B chain peptides.

EXAMPLE 3 Receptor Competition Assays

Assays were performed using confluent cultures of NIH 3T3 cells in 24well plates (Costar) grown in DMEM, 10% fetal calf serum, 10 μg/mlGentamicin. The growth media was removed and the cells washed twice withserum-free DMEM, 0.2 mg/ml BSA and the plates placed on ice for 30minutes in serum-free DMEM, 0.2 mg/ml BSA. Test samples and controlswere made up in serum-free DMEM, 0.2 mg/ml BSA containing 5-10 ng/ml ofHUVE affinity purified proteins and a serial dilution of one of therecombinant PDGF isoforms in a concentration range of 300 ng/ml to 16ng/ml. One milliliter aliquots of the samples were placed into wells ofthe 24 well plates and incubated on ice on a platform rocker for twohours. After the incubation period, the cells were washed three timesfor 10 minutes each on ice with PBS. The proteins bound to the surfaceof the cells were eluted with 5 ul of 1 N acetic acid for 10 minutes.The acetic acid elution samples were lyophilized, resuspended in 5 mMHCL, run on 12% polyacrylamide gels and immunoblotted to nitrocelluloseusing the anti-PDGF antibody.

In order to substantiate the binding of the endothelial cell moleculesto the PDGF cell surface receptors, competitive receptor binding assayswere performed. Because immunoblots of the affinity purified HUVE cellsecreted proteins indicated the presence of multiple PDGF immunoreactivemolecules, ¹²⁵ I-labeled PDGF competition assays could not be used sincethis would not indicate which molecules in this mixture were competingfor binding of the labeled PDGF for the receptors on the target cells.Since the isoforms of PDGF and the major PDGF immunorelated proteinsecreted by HUVE cells are of different molecular weights, receptorbinding competition was demonstrated on immunoblots. Direct binding ofthe anti-PDGF immunoreactive peptides to NIH 3T3 cells was demonstratedby incubating monolayers of the 3T3 fibroblasts with the anti-PDGFaffinity purified proteins (10 ng/ml) for 2 hours at 4° C. Boundpeptides were released by washing of the cell layer with 1 N acetic acidand quantitated by immunoblot analysis using anti-PDGF IgG. This datashow that the 36 kD immunoreactive peptide binds to cell surface of NIH3T3 cells. This binding can be competed by increasing concentrations ofrecombinant PDGF BB added to the binding media. These data suggest thatthe CTGF peptide binds to specific cell surface receptors on NIH 3T3cells and that PDGF BB can compete with this binding.

RNA Isolation and Northern Blotting

Total RNA was isolated from cells in monolayer culture cells.Lyophilized RNA was resuspended in gel loading buffer containing 50%formamide and heated at 95° C. for two minutes before loading (20 μg perlane total RNA) onto 2.2 M formaldehyde, 1% agarose gels and run at 50volts. Integrity of RNA was determined by ethidium bromide staining andvisualization of 18S and 28S rRNA bands. After electrophoresis the RNAwas transferred to nitrocellulose by blotting overnight with 10× SSCbuffer. The nitrocellulose was air dried and baked at 80° C. for 2 hoursin a vacuum oven. Hybridization was performed overnight at 46° C. withthe addition of 5×10⁵ CPM per ml of ³² P-labeled probe. Normally forNorthern blots, the entire plasmid was labeled and used as a probe.Labeling was done with a random primer labeling kit from BoehringerMannheim. After hybridization, membranes were washed twice in 2× SSC,0.1% SDS for 15 minutes each at room temperature, once for 15 minutes in0.1× SSC, 0.1% SDS, room temperature and a final 15 minutes wash in 0.1×SSC, 0.1% SDS at 46° C. Blots were autoradiographed at -70° C. on KodakX-omat film.

EXAMPLE 4 Library Screening, Cloning, and Sequencing

Standard molecular biology techniques were used to subclone and purifythe various DNA clones (Sambrook, et al., Molecular Cloning a LaboratoryManual, Second edition, Cold Spring Harbor Laboratory Press, Col. SpringHarbor, N.Y.). Clone DB60 was picked from a lambda gt11 HUVE cell cDNAlibrary by induction of the fusion proteins and screening with anti-PDGFantibody. Plaques picked were rescreened and positive clones replated atlow titer and isolated.

The EcoR I insert from clone DB60 was cloned into the M13 phage vectorand single-stranded DNA obtained for clones with the insert in oppositeorientations. These M13 clones were then sequenced by the dideoxy methodusing the Sequenase kit (U.S. Biochemical) and ³⁵ S-dATP (duPont). Bothstrands of DNA for this clone were completely sequenced using primerextension and both GTP and ITP chemistry. Aliquots of the sequencingreactions were run on both 6% acrylamide (16 hours) and 8% acrylamide (6hours) gels, vacuum dried and autoradiographed for at least 18 hours.

The cDNA fragment from clone DB60 was ³² P-CTP labeled and used torescreen the HUVE cell cDNA lambda gt11 library. Several clones werepicked and the largest, the 2100 bp clone designed DB60R32, wassubcloned into Bluescript phagemid. Subclones were made of Pst I, Kpn I,and Eco RI/Kpn I restriction fragments also in Bluescript. Thesesubclones were sequenced by double-stranded plasmid DNA sequencingtechniques using Sequenase as described above. The 1458 bp Eco RI/Kpn Iclone containing the open reading frame was subcloned into M13 mp18 andM13 mp19 and both strands of DNA were completely sequenced usingsingle-stranded DNA sequencing techniques with primer extension and bothGTP and ITP chemistry.

Cloning Expression and Sequencing of the cDNA for Connective TissueGrowth Factor

In order to further characterize these PDGF related molecules,sufficient quantities of the CTGF protein for amino acid sequencing wasneeded. However, the low concentrations of CTGF in the conditioned mediaof HUVE cell cultures and the costly and time consuming techniquesinvolved in obtaining and culturing these cells made proteinpurification to homogeneity and amino acid sequencing impractical.Therefore, the anti-PDGF antibody was used to screen an HUVE cell cDNAlibrary made in the expression vector lambda gt11. Over 500,000recombinant clones were screened. Several clones which gave strongsignals with the anti-PDGF antibody in the screening process werepurified and subcloned into the M13 phage vector and partial sequencedata obtained by single-stranded DNA sequencing. A search of the GenBankDNA sequence data base indicated that two of the clones picked containedfragments of the PDGF B chain cDNA open reading frame sequence. One ofthese clones was similar to a 1.8 kb insert previously isolated byCollins, et al. (Nature, 316:748-750, 1985) using a c-sis cDNA probe. Athird clone of 500 bp was completely sequenced and no match was found ina homology search of all nucleotide and amino acid sequences in GenBank(CEF 10 sequence was not available at that time). This clone wasdesignated DB60. Anti-PDGF antibody binding to the fusion proteinproduced by the clone DB60 was completely blocked by the affinitypurified proteins. A ³² P-labeled probe was made of DB60 and used on aNorthern blot of 20 μg of total RNA isolated from HUVE cells. The blotindicated probe hybridization with an mRNA of 2.4 kilobases, which is amessage of sufficient size to produce the proteins in the 38 kDmolecular weight range seen on the immunoblots of the affinity purifiedproteins. The DB60 clone was used to rescreen the HUVE cell cDNA lambdagt11 library and the largest clone isolated contained a 2100 base pairinsert designated DB60R32. A probe made with the 2100 bp Eco RI insertof clone DB60R32 also hybridized with a single 2.4 kb message in aNorthern blot of total RNA from HUVE cells.

EXAMPLE 5 In Vivo Transcription and Translation

In vitro transcription reactions were done using the 2100 bp cDNA cloneDB60R32 in the Bluescript KS vector. The plasmid was cut with Xho Iwhich cuts the plasmid once in the multiple cloning site of the vector3' to the cDNA insert. The T7 promoter site located 5' to the cDNAinsert was used for transcription. The in vitro transcriptions were donewith a kit supplied with the Bluescript vector (Stratagene).

In vitro translation reactions were done using nuclease treated rabbitreticulocyte lysate and ³⁵ S-cysteine in a cysteine-free amino acid mixfor labeling of the peptide (Promega). The reactions were done in afinal volume of 50 ul containing ³⁵ S-cysteine 1 mCi/ml (1200 Ci/mMole,DuPont), and serial dilutions of mRNA from the in vitro transcriptionreactions in concentrations ranging from 50 to 500 nanograms perreaction tube. The reactions were incubated at 30° C. for 60 minutes.Aliquots of the reactions were run on reduced or nonreduced 12%polyacrylamide electrophoresis gels, dried, and autoradiographed.

Bacterial expression of immunoreactive CTGF peptide was accomplished bysubcloning clone DB60R32 into the Eco RI site of the pET 5 expressionvector (Studier, et al., Ed. Academic Press, New York Vol. 185, 60-89,1990) in both sense and inverse orientations (as determined byrestriction enzyme digest analysis). Cultures of E. coli HMS174 cellswere grown in M9 media to an OD 600 of 0.7 and the media made 0.4 mMIPTG and incubation continued for 2 hours. The cells were pelleted,lysed, inclusion bodies removed by centrifugation and aliquots of thepellet extracts run on 12% polyacrylamide gels and immunoblotted usingthe anti-PDGF antibody. The protein produced by clone DB60R32 in thesense orientation produced anti-PDGF immunoreactive peptides in the36-39 kD MW range while the anti-sense control produced noimmunoreactive peptides. The recombinant peptides produced in the E.coli system completely blocked the anti-PDGF reaction with the CTGFpeptides present in conditioned media.

Expression of CTGF in Xenopus

For expression in Xenopus oocytes, mature X. laevis females wereobtained from Nasco (Fort Atkinson, Wis.) and maintained at roomtemperature. Frogs were anesthetized by hypothermia and the ovariantissue was surgically removed. Ovarian tissue was minced and digestedthe 0.2% collagenase (Sigma Type II) in OR-2 without calcium (Wallace,et al., Exp. Zool., 184:321-334, 1973) for 2-3 hours. Unblemished stageVI oocytes (Dumont, J. Morphol., 136:153-180, 1972), 1.3 mm diameter,were then carefully selected and microinjected.

Stage VI oocytes (5-10 at a time) were placed on a hollowed plexiglassplatform and drained of excess OR-2 solution. Approximately 50 nl ofsample containing 10 ng of RNA was injected into the animal pole justabove the oocyte equator using a Leitz system microinjector. Followinginjection, oocytes were returned to OR-2 buffer with 0.1% BSA andincubated for 24 hours at 25° C. Viable oocytes were then pooled andextracted by homogenization in 100 mm NaCl, 10 mm Tris pH 7.5 with tenstrokes of a Dounce homogenizer (20 μl/oocyte). The homogenate was thenmixed with an equal volume of freon to remove pigment and lipid andcentrifuged at 10,000 rpm for 30 seconds to separate the phases. The topaqueous phase was removed and tested for chemotactic activity using NIH3T3 cells as described above.

Injection of Xenopus oocytes with 10 ng of RNA preparations derived byin in vitro transcription of the DB60 R32 clone resulted in theproduction of a fibroblast chemotactic activity. Control injected cellsdid not produce this activity. These results indicate that the openreading frame of the DB60 R32 clone encodes a protein with chemotacticactivity for fibroblastic cells as does CTGF.

EXAMPLE 6 Sequence Analysis of CTGF

The 2100 bp insert of clone DB60R32 was sequenced initially bysubcloning of Pst I and Kpn I restriction fragments into Bluescript andusing double-stranded dideoxy methods. This indicated an open readingframe of 1047 base pairs and oriented the DB60 insert to the largercDNA. An Eco RI/Kpn I fragment containing the entire open reading framewas inserted into M13 mp18 and M13 mp19 and both strands of the DNA weresequenced with single-stranded dideoxy methods by primer extension usingboth GTP and the GTP analog ITP. The cDNA nucleotide sequence of theopen reading frame encoded a 38,000 MW protein, confirming the cell-freetranslation results and matching the size of the immunopurifiedpeptides. A new search of the GenBank data base revealed that this cDNAhad a 50% nucleotide sequence homology with CEF-10 mRNA, one of theimmediate early genes induced in v-src transformed chicken embryofibroblasts (Simmons, et al., Proc. Natl. Acad. Sci. USA, 86:1178-1182,1989). The translated cDNA for human CTGF and avian CEF-10 have a 45%overall homology and a 52% homology if the putative alternative splicingregion is deleted. This region is between amino acids 171 (asparticacid) and 199 (cysteine) in the CTGF sequence.

EXAMPLE 7 Analysis of CTGF Promoter Region

Cell Cultures

Human skin fibroblasts were grown from explants of skin biopsyspecimens. NIH/3T3 cells and Cos 7 cells were obtained from the AmericanType Culture Collection (ATCC, Rockville, Md.) All cells were culturedin Dulbecco's modified eagle's medium (DMEM) contained 10% fetal calfserum (FCS) at 37° C. in an atmosphere of 10% CO₂ and 90% air. Humanskin fibroblasts were used prior to the sixth passage.

Growth Factors

TGF-β 1 was a gift from Richard Assoian (U. Of Miami). Recombinant PDGFBB was obtained from Chiron (Emeryville, Calif.). Purified murine EGFwas purchased from Sigma (St. Louis, Mo.).

RNA Isolation and Northern Blotting

Total RNA was isolated from cultured cells by acid guanidiumthiocyanate-phenol-chloroform extraction as reported previously(Chomczynski, et al., Birchem, 162: 156-159, 1987). Total RNA waselectrophoresed on an 1.5% agarose/formaldehyde gel and transferred tonitrocellulose. The CTGF probe as 1.1 kb fragment representing the CTGFopen reading frame obtained by PCR reaction using specific primers HO15'-CGGAATTCGCAGTGCCAACCATGACC-3' (SEQ ID NO:3) and HO25'-CCGAATTCTTAATGTCTCTCACTCTC-3' (SEQ ID NO:4). Hybridizations wereperformed using 1×10⁶ cpm/ml of these probes labeled with [³² α-P]dCTPby using a Random Primer DNA Labeling Kit (Boehringer MannheimBiochemicals, Indianapolis, Inc.). Autoradiography was performed at -70°C. for 6 to 72 hours by using X-ray films and intensifying screens.

Isolation of Genomic Clones and Sequence Analysis

Genomic DNA was isolated from human skin fibroblasts as describedpreviously (Sambrook, et al., Cold Spring Harbor Laboratory Press, 9:14-19, 1989.). Using 4 μg of genomic DNA as a template, a fragment ofthe CTGF gene was amplified by PCR using primers HO2 and HO35'-CGGAATTCCTGGAAGACACGTTTGGC-3' (SEQ ID NO:5). PCR products weredigested with EcoRI and subcloned into M13. Sequence analysis by thedideoxy chain termination method (Sanger, et al., Proc. Natl.Acad.Sci.USA, 74: 5463-5467, 1977.) Using the Sequenase kit *U.S. BiochemicalCorp., Cleveland, Ohio) demonstrated 900 bp fragment which had a 387 bpintern in the middle portion. Using a Human Genomic Library in theLambda FIX™ II vector (Stratagene, La Jolla, Calif.) we screenedapproximately 1×10⁶ recombinant phages with ³² P-labeled 900 bp genomicDNA fragment as probe and isolated 3 phage clones that contained theCTGF gene.

Luciferase Reporter Gene Assays

A fragment of the CTGF promoter containing nucleotides -823 to +74 fromone of the human genomic clones was first cloned in the Sac1-Xho1cloning site of pGL2-Basic vector (Promega). This construct (PO) wasused as a template for PCR and deletion mutants were made with specificprimers as follows: P1 contained nucleotides from -638 to +74, P2 from-363 to +74, P3 from -276 to +74, and P4 from -128 to +74. All deletionfragments were sequenced to insure no mutations had been introduced inthe promoter fragments. NIH/3T3 cells were transfected in a 6-well platewith LIPOFECTIN® reagent (GIBCO BRL) for 6 hours. Each transfectionincluded 2 μg of pSV-β-Galactosidase vector (Promega). Cells wereincubated in serum-free DMEM with ITS™ (Collaborative BiomedicalProducts) for 24 hours after transfection followed by the incubationwith growth factors for 4 hours or 24 hours. Luciferase activity wasmeasured by using Luciferase Assay System (Promega) and a scintillationcounter (Beckman LS6000SC) using it in single photon monitor mode. Tonormalize for differences in transfection efficiency β-galactosidaseactivity was measured using a chemiluminescent assay usingGalacto-Light™ (TROPIX, Inc.).

Preparation of Nuclear Extracts

Nuclear extracts were prepared as described by Abmayr and Workman(Current Protocols in Molecular Biology, vol 2, pp12.1.1-12.1.9,Ausubel, et al., Greene Publ., and Wiley lnterscience, New York, N.Y.Briefly, cells were treated with hypotonic buffer (10 mM HEPES pH 7.9,1.5 mM MgCl₂, 10 mM KCl, 0.2 mM PMSF, 0.5 mMDTT), homogenized with 10strokes of a glass dounce homogenizer and nuclei were isolated bycentrifugation at 3300×g for 15 minutes. Nuclear proteins were extractedby suspending the nuclei in an equal volume of extraction buffer (20 mMHEPES pH7.9, 25% glycerol, 1.5 mM MgCl₂, 0.8M KCl, 0.2 mM EDTA, 0.2 mMPMSF and 0.5 mM DTT). The extract w as dialyzed against 20 mM HEPES pH7.9, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.2 mM PMSF and 0.5 mM DTTbefore use. Protein concentration was determined using the BCA proteinassay regent (Pierce).

Gel Mobility Shift Assay

Fragments of the CTGF promoter were prepared by PCR or restrictionendonuclease digestion of the promoter fragment. Double strandedoligonucleotides were prepared by annealing complementary singlestranded oligonucleotides. All oligonucleotides and fragments werechecked by electrophoresis in agarose gels or polyacrylamide gels.Radiolabeled fragments of the CTGF promoter were prepared byend-labeling with Klenow enzyme (Boehringer Manheim) and polynucleotidekinase (Boebringer Manheim). Labeled fragments were purified byelectrophoresis in 2% agarose gels or 20% polyacrylamide gel before usein gel mobility shift assay. The binding reaction mixture contained 1 μgof nuclear extract protein in 20 μl of 10 mM HEPES pH 7.9, 5 mM Tris, 50mM KCl, 0.1 mM EDTA, 1 μg poly(dl-dC)poly(dl-dC) (Phamacia), 10%glycerol, 300 μg/ml BSA, and 10,000 cpm ³² P labeled DNA probe.Unlabeled competitor DNA was added and incubated at 4° C. for 2 hoursprior to adding the labeled probe. The labeled probe was incubated forone hour at 4° C. in the reaction mixture. Electrophoresis was performedusing 5% polyacrylamide gel with 50 mM Tris, 0.38M glycine and 2 mMEDTA.

Methylation Interference Assay

End-labeled fragments of double stranded oligonucleotides were preparedas described for the gel mobility shift assay. The oligonucleotides weremethylated by dimethyl sulfate (Fisher Scientific) for 5 minutes at roomtemperature. DNA-protein binding and gel mobility shift assay wereperformed as described above using large amounts of labeled probe (100Kcpm) and nuclear protein (20 μg). DNA from shifted and non-shifted bandswas purified and cleaved with piperidine (Fisher Scientific), and thesamples were electrophoresed on a polyacrylamide DNA sequencing gel. Thesequences of the shifted and non-shifted fragments were compared withthe intact probe sequenced using the same methods.

EXAMPLE 8 Prolonged Induction of CTGF mRNA by Short Term TGF-β Exposure

Most immediate early genes, such as c-fos and c-myc, that are induced bygrowth factors exhibit a short burst of expression even though thegrowth factor remains present in the media. In contrast, CTGFtranscripts remain at high levels for over 24 hours after activation ofthe cells with TGF-β (Igarashi, et al., Mol. Biol Cell, 4: 637-645,1993). This example examines whether the long term elevation of CTGFtranscripts was dependent on the continuous presence of TGF-β.

Confluent human skin fibroblasts were cultured in serum free DMEMsupplemented by insulin, transferrin, and selenium (DMEM-ITS) for 24hours prior to adding of TGF-β. After 1 hour exposure to TGF-β, cellswere washed in PBS and replaced with DMEM-ITS followed by differentperiods of incubation. Specifically, confluent cultures of human skinfibroblasts were incubated with DMEM-ITS containing 5 μg/ml of insulin,5 μg/ml of transferrin and 5 ng/ml of selenium for 24 hours prior to theaddition of TGF-β. After the treatment with 10 ng/ml of TGF-β for 1hour, cells were washed with PBS and incubated with DMEM-ITS forindicated time periods. Northern blot analysis revealed CTGF mRNA wasstrongly induced from 4 hours to 30 hours after TGF-β removal (FIG. 2A).

The ability of TGF-β to induce the CTGF transcript in the presence ofseveral protein synthesis inhibitors was examined. FIG. 2B shows theeffect of cycloheximide on induction of CTGF mRNA. Lane A and H arenon-treated control cells at 4 hours and 24 hours, respectedly. Lane B,4 hrs. Cycloheximide (CHX) (10 ug/ml); Lane C, 4 hrs TGF-β present for 1hour during hour 1 of 2 of cycloheximide exposure; Lane E, same as Bwith RNA prepared 24 hours after addition of cycloheximide; Lane f, 24hours TGF-β (10 ug/ml); Lane G, same as D with RNA prepared 24 hoursafter addition of cycloheximide and 22 hours after removal of TGF-β.

As shown in FIG. 2B, a 1 hour stimulation by TGF-β in the presence ofcycloheximide was sufficient to induce CTGF mRNA 4 hours later as wellas 24 hours later. Cycloheximide alone was able to increase CTGF mRNA 4hours later, suggesting the possibility of mRNA stabilization as hasbeen reported for cycloheximide induction of other transcripts such asc-fos and c-myc (Greenberg, et al., Nature (London), 311: 433-438, 1984;Kruijer, et al., Nature, 312: 711-716, 1984). However, a report byEdwards and Mahadevan (Edwards, D. R., and L. C. Mahadevan, EMBO J,11:2415-2424, 1992) indicated that the protein synthesis inhibitorscycloheximide and anisomycin, but not puromycin, could act to stimulatetransciption of the c-fos and c-jun genes, therefore messagestabilization is not the only possible mechanism of action for thesecompounds.

The ability of anisomycin and puromycin to inhibit TGF-β induction ofCTGF transcripts was compared with that of cycloheximide as to theability to elevate CTGF transcripts. FIG. 2C shows the effect of proteinsynthesis inhibitors on induction of CTGF mRNA. Cells were treated withpuromycin or anisomycin for 4 hours. TGF-β was added 1 hour after theaddition of protein synthesis inhibitor and cells were incubated for 3hours prior to isolation of total RNA. CTGF transcripts were analyzed bynorthern blot.

Puromycin did not induce the CTGF mRNA at any of the concentrationstested up to 100 μg/ml, which is 10 fold higher than that needed tocompletely block protein synthesis in these cells. Even at this highconcentration it had no effect on the ability of TGF-β to induce theCTGF mRNA. In contrast, anisomycin did elevate CTGF transcripts (FIG.2C) as was seen with cycloheximide although TGF-β treatment still raisedthe level of CTGF mRNA in the presence of anisomycin.

These findings are similar to those reported by Edwards and Mahadevan(Edwards, D. R., and L. C. Mahadevan., EMBO J, 11:2415-2424, 1992) whereboth c-fos and c-jun were induced by anisomycin or cycloheximide bythemselves, but not by puromycin alone. These data strongly suggestTGF-β directly regulates CTGF gene expression via a mechanism that isindependent of protein synthesis and may be primarily acting at thelevel of transcription.

EXAMPLE 9 Isolation of the Human CTGF Gene

To elucidate the structure of the CTGF gene, a fragment of the CTGF genewas first obtained using PCR. Four micrograms of genomic DNA preparedfrom human skin fibroblasts was used as a template and oligonucleotides,HO2 and HO3, were used as primers. After 30 cycles of reaction, a 900 bpfragment was recovered that was 390 bp longer than predicted from thecDNA sequence. Nucleotide sequence analysis of this fragment (HO900)revealed the presence of a 387 bp intron in the middle portion of thefragment. Using HO900 as a probe, 3 phage clones were from the humangenomic library that contained a 4.3 kb Xba1 fragment which representedthe entire coding sequence of the CTGF gene and a large portion of theputative promoter region. As shown in FIG. 1A, the CTGF gene has 5 exonsand 4 introns. A TATA sequence is present 24 nucleotides upstream of themRNA cap site, determined by oligonucleotide primer extension. Theconsensus sequence of a CArG box, which is the inner core of the serumresponse element (SRE) characterized by CC(A/T)₆ GG, is present betweennucleotide position -380 to -390. Other potential regulatory elementsare also present including a CAT box, two Sp1 sites and two AP-1 sites.Furthermore, the CTGF promoter has a NF-1 like site, (TGGN₆ GCCAA) (SEQID NO:6), between positions -194 and -182, and TGF-β inhibitory elementlike sequence (GNNTTGGTGA) (SEQ ID NO:7) between positions -119 and-128. Both of these elements have single base differences from thereported consensus sequences (Edwards, D. R., and J. K. Heath, Thehormonal control regulation of gene transcription, 16: pp 333-347) DNAsequence comparison showed that the human CTGF promoter has an 80%sequence identity to the murine fisp-12 promoter in the region 300nucleotides 5' of the transcription start site (FIG. 1B). Furtherupstream regions exhibit much less similarity in DNA sequence.

EXAMPLE 10 Studies on the CTGF Promoter

To test whether the 5' nontranslated region of CTGF gene functions as aTGF-β inducible promoter, a fusion gene was constructed containing theCTGF promoter (nucleotides -823 to +74; nucleotides 1 to 897, SEQ IDNO: 1) and the coding region of the firefly luciferase gene in thevector pGL2-basic. Luciferase activity was tested in a transienttransfection assay using NIH/3T3 cells. This construct conferred a 15-30fold induction of luciferase activity after 24-hour stimulation by TGF-βcompared with control cultures. As seen at the level of the CTGF mRNA,other growth factors such as PDGF, EGF and FGF stimulated only a 2-3fold induction of luciferase activity under identical conditions (Table1).

                  TABLE 1                                                         ______________________________________                                        CELL TYPE AND GROWTH FACTOR REGULATION                                         OF THE CTGF PROMOTER                                                          RELATIVE FOLD INDUCTION OF LUCIFERASE ACTIVITY                                AFTER GROWTH FACTOR TREATMENT                                                    CELL TYPE   TGF-β                                                                            PDGF     FGF  EGF                                     ______________________________________                                        NIH/3T3     25.7    2.9        3.3  1.4                                         HSF 9.2 2.4 3.1 2.2                                                           VSMC 9.8 ND ND ND                                                             HBL 100 1.1 ND 1.3 1.4                                                        HEP G2 1.3 ND 1.4 1.8                                                       ______________________________________                                         ND Not Determined                                                             HSFHuman foreskin fibroblasts (primary)                                       VSMCFetal bovine aortic smooth muscle cells (primary)                         HBL 100 Human breast epithelial cell line (nontumorigenic)                    HEP G2 Human Hepatic epithelial cell line (nontumorigenic)                    (A CTGF gene fragment extending from nucleotides position 823 to +74 was      inserted in the pGL2basic vector. Plasmids were transfected with              lipofectin for 6 hours and cells were incubated in DMEMITS for 16 hours       prior to the addition of growth factors. After a 24 hour incubation cell      extracts were prepared and luciferase activity measured. Luciferase           activities were normalized by measuring galactosidase activity  #             expressed from a cotransfected lacZ expression vector, pSVgalactosidase       vector and compared between growth factor treated cells and nontreated        cells. These experiments were repeated 4 times with similar observations.     A representative experiment is shown).                                   

When this promoter fragment was cloned in the reverse orientation (+74to -823), only basal levels of luciferase activity were detected andthis level was uneffected by TGF-β or other growth factor treatment ofthe cells. The same pattern of growth factor induction was observed whenhuman skin fibroblasts were used instead of NIH/3T3 cells (Table 1).TGF-β did not induce luciferase activity in several epithelial celllines (Table 1), demonstrating that TGF-β regulation of the CTGF gene iscell type specific. The lack of any response by the epithelial cells isnot due to a lack of a TGF-β response as the growth of these cells isinhibited by TGF-β (10 ng/ml). The induction of luciferase activityunder the control of the CTGF promoter only required a brief exposure ofthe cells to TGF-β as a 1 hour treatment of the cells with TGF-β givesnearly the same fold induction at 4 and 24 hours as cells continuouslyexposed to TGF-β (Table 2). These results confirm the data from theNorthern blots described previously and demonstrate that transcriptionalregulation plays a primary role in the control of CTGF gene expressionby TGF-β.

                  TABLE 2                                                         ______________________________________                                        SHORT TERM TGF-B EXPOSURE STIMULATES                                            LONG TERM CTGF PROMOTER ACTIVITY.sup.1                                                     Time of Assay of Luciferase Activity                           Duration of TGF-β exposure                                                              4 Hours      24 Hours                                          ______________________________________                                        Continuous     3.8          21                                                  1 hour 3.5 19                                                               ______________________________________                                         .sup.1 Fold induction of Luciferase activity determined as described in       Table 1 legend and EXAMPLE 7. NIH/3T3 cells were used for these               experiments.                                                             

EXAMPLE 11 Identification of the Promoter Element Required for TGF-βInduction

To determine which region of the promoter sequence is responsible forthe induction by TGF-β, deletion mutants of the CTGF promoter wereconstructed using PCR primers designed to delete the known transcriptionfactor consensus elements. Regions of the promoter beginning at the most5' region and moving toward the transcription start site weresystematically deleted (FIG. 3A). Removal of the region of the promoterdown to base -363 which included an AP1 site and the CArG box had nosignificant effect on the TGF-β induction of luciferase activity.Approximately a 30% reduction was seen when the second AP-1 was deleted(-363 to -276) although the fold induction by TGF-βwas still high (20fold). Removal of the NF-1 like site in the P4 construct (-276 to -128)eliminated the TGF-β inducibility of the promoter suggesting that thisregion contained the TGF-β response element. Taking advantage of twoBsmI sites we deleted the nucleotides from -162 to -1 10 leaving theremaining portions of the promoter intact. This construct exhibited acomplete loss of TGF-β inducibility demonstrating that the sequencebetween positions -162 and -128 is essential for the TGF-β induction ofluciferase activity. This region contains the TGF-β inhibtory element(TIC)-like site and is bordered by the NF-1 like site that others havereported plays a role in TGF-β regulation of α2(I) collagen geneexpression (Oikarinen, J., A. Hatamochi, and B. De Crombrugghe., J.Biol. Chem. 262:11064-11070, 1987.) And type 1 plaminogen activatorinhibitor (PAI-1) gene expression (Riccio, et al., Mol. Cell Biol,12:1846-1855, 1992.).

A fusion gene was constructed placing the nucleotides from positions-275 to -106 of the CTGF promoter upstream from an SV40 enhancerlesspromoter controlling a luciferase gene to determine whether this regionof the promoter was sufficient to confer TGF-β inducibility (FIG. 3B).The SV40 enhancerless promoter was not regulated by TGF-β. However, thepromoter containing the CTGF sequences -275 to -106 conferred a nearly 9fold induction after TGF-β treatment. Inversion of the fragment resultedin a little stimulation of luciferase activity after TGF-β treatment.These data confirm that sites in the CTGF promoter between nucleotides-275 and -106 can act as TGF-β regulator elements.

A series of competitive gel shift and methlylation interference assayswere performed to delineate the region of this potion of the CTGFpromoter that was binding to nuclear proteins. Initially competitive gelshifts were used to delineate which region of the sequence betweenpositions -205 to -109 was a target for protein binding. A diagram ofthe probe and the various competitor fragments is illustrated in (FIG.4). The results of these studies demonstrate that any fragment thatcontained the NH3 region (-169 to -149) acted as a specific competitorfor the labeled promoter fragment containing bases -204 to -109 (FIG.4). This region is located between the NF-1 like and TIE like sites.Oligonucleotide fragments that contained only the TIE like region or theNF-1 like region without the NH3 region did not compete in the gel shiftassay.

To further delineate the regulatory element, methylation interferenceassays were performed. A fragment of the promoter from positions -275 to-106 was used initially. The results of these studies indicate thatneither the NF-1 like site of the TIE element appear to be interactingwith any nuclear proteins present in either control or TGF-β treatedcells confirming the gel shift competition data. However, a regionbetween these sites from positions -157 to -145 (nucleotides 669 to 681;SEQ ID NO: 1) contained several G residues that were not methylated inthe shifted bands suggesting that this region was the nuclear proteinbinding site (FIG. 5A). A smaller fragment of the region (nucleotides-169 to -139) was then analyzed to give better resolution of theimportant G residues (FIG. 5B). The data from this analysis confirmedthat of the larger fragment and map G residues that lie within thesequence determined by competition gel shifts.

To better characterize the actual TGF-β reactive site, the ability ofthe intact sequence and several deletions to compete for protein bindingwas compared in the gel shift assay (FIG. 6). These data confirm theresults of the methlyation interference assays and suggest that theregion of the promoter from positions -159 to -143 contains at least aportion of the cis regulator element involved in TGF-β regulation ofCTGF gene expression.

Point mutations were made in the region of the sequence believed to beinvolved in the TGF-β induction and tested these promoters in ourluciferase reporter construct (FIG. 7). Two point mutations were testedand both reduced the inducibility of the gene by TGF-β. One mutationreduced the induction by 25% from control and the other by 80%. Neitherhad any effect on basal level of expression compared to control nativesequence.

Point mutations were constructed by synthesis of oligonucleotidescontaining the desired base change and taking advantage of the two BsmIsites in the CTGF promoter. All constructs were confirmed by nucleotidesequence analysis to demonstrate that only the desired base changeoccurred and that all of the other nucleotide sequence was identical tothe normal promoter. Assays were performed as described above for otherCTGF promoter-luciferase constructs using NIH/3T3 cells as targets. Thedata presented in the Table in FIG. 7 is from a single experiment withduplicate assays for each experimental condition. The experiment was runseveral times to confirm the results. These data demonstrate that asingle mutation in this region of the promoter can reduce the TGF-βinduction by 85%, to less than 15% of the normal gene. These datademonstrate that the sequence identified is essential for the TGF-βinduction of the CTGF gene.

EXAMPLE 12 TGF-β Stimulates Anchorage Independent Growth Via a CTGFDependent Pathway

a. Inhibition of TGF-β Induced CTGF Gene Expression by Elevation of cAMPLevels

Both herbimycin and phorbol esters were utilized to determine if eithertyrosine kinases or protein kinase C had any role in the regulation ofCTGF gene expression induced by TGF-β. These studies were performedusing the CTGF promoter (-823 to +74) luciferase reporter constructtransfected into NIH/3T3 cells.

NIH/3T3 cells were grown to 50% confluence in DMEM/10% FCS. They wereall transfected with PO CTGF promoter (nucleotides position -823 to +74)driving the expression of the pGL2 basic vector firefly luciferase usingLIPOFECTIN® as described above in EXAMPLE 7. After 24 hours in DMEM/ITSmedia the inhibitors were added. All of the agents were added to thecultures 2 hours prior to the addition of 10 ng/ml TGF-β. The cells wereincubated 24 hours and the luciferase activity determined using theTropix luciferase assay kit and a Beckman scintillation counter equippedwith a single photon monitor. In a related experiment PMA was added tothe cells for 24 hours prior to TGF-β to deplete protein kinase C. Thisalso had no effect on the ability of TGF-β to induce luciferase activityunder the control of the CTGF promoter. Also, as a control experimentfor the herbimycin studies, the activity of this agent to inhibit PDGFinduced cell division was examined. Confluent density arrestedmonolayers of NIH/3T3 cells were treated with the indicatedconcentrations of herbimycin for 2 hours prior to addition ofrecombinant PDGF BB. The number of cells was determined bytrypsinization and counting 24 hours after the addition of PDGF. Themitotic index represents the percent of cells that underwent mitosis.

Neither of these compounds had any effect on the ability of TGF-β toinduce CTGF gene expression, nor did they modulate the basal level ofCTGF gene expression in the target cells. However, both cholera toxin or8Br-cAMP were potent inhibitors of the TGF-β induction of the CTGF gene(FIG. 8A).

These data indicate that neither tyrosine kinases or protein kinase C ispart of the signal transduction pathway leading to CTGF gene inductioncontrolled by TGF-β. Also, cyclic nucleotide regulated proteins do notappear to be a part of the TGF-β pathway for regulation of CTGF geneexpression. However, elevation of cAMP levels in the cell abolishes theTGF-β induction of CTGF gene expression. In a related experiment we findthat the cAMP or cholera toxin can be added up 8 hours after addition ofTGF-β and it is still effective in blocking the expression of the CTGFgene. This suggests that the action of the cAMP is distal from thereceptor and may be effecting transcription factor binding to the CTGFpromoter.

b. cAMP Does Not Block All of the Actions of the TGF-β on FibroblastCells

The four panels shown in FIG. 8B are photomicrographs of the NIH/3T3cells used in the above experiments which were Control) No Additons;TGF-β) TGF-β (10 ng/ml); cAMP) 8Br cAMP (1000 uM); cAMP+TGF-β) 8Br cAMP(1000 uM) and TGF-β (10 ng/ml) prior to determination of luciferaseactivity. These data indicate that while cAMP causes dramatic changes inthe morphological appearance of the NIH/3T3 cells, TGF-β addition tothese cells induces a morphological appearance in these cells which wassimilar, if not identical, to control cells treated with TGF-β. Thus,although cAMP can block TGF-β induction of CTGF gene expression it hasno effect on the biochemical events which regulate the observed changesin morphology seen in these monolayer cultures. These resultsdemonstrate that there are multiple components in the action of TGF-β onfibroblastic cells which can be differentially blocked by cAMP. Nosignificant difference was detected in the culture with respect to totalcellular protein content or expression of an SV40/β-galactosidasecontrol reporter gene indicating that the changes were not due to toxiceffects of the AMP. Cholera toxin treatment induced a morphology similarto that seen in the 8Br cAMP treated cells which was reversed byaddition of TGF-β.

c. Inhibition of TGF-βInduced Anchorage Independent Growth by cAMP andIts Reversal by rCTGF

Because of the results of the previous studies, experiments wereperformed to determine whether cAMP would block TGF-β induced anchorageindependent growth. Initially the effects of 8Br cAMP, 8Br cGMP andcholera toxin on the ability of TGF-β to induce anchorage independentgrowth of NRK cells was examined. Anchorage independent growth assayswere performed essentially as described by Guadagna and Assoian (J CellBiol, 115: 1419-1425, 1991). Briefly NRK cells normally maintained asmonolayer cultures were plated on an agarose layer in DMEM/10% FCScontaining 5 ng/ml EGF. TGF-β or CTGF was added and the cells incubatedfor 72 hours. DNA synthesis is then determined by labeling for 24 hourswith ³ H-thymindine (2 uCi/ml) the cells harvested and processed by TCAprecipitation etc. Inhibitors were added at the same time as the growthfactors and remained present for the duration of the experiment (FIG.8C). (Abbreviations: Cholera toxin (CTX)).

As seen in FIG. 8C both 8Br cAMP and cholera toxin were effectiveinhibitors of growth in this assay while 8Br cGMP had no effect atconcentrations up to 10 mM. Because expression of the CTGF gene wasblocked by elevation of cAMP levels in the cells, an experiment wasperformed to determine whether rCTGF could overcome the inhibition. Asseen in the left panel in FIG. 8B addition of rCTGF to NRK cells doesnot stimulate anchorage independent growth and therefore does notsubstitute for TGF-β. However, addition of the same amounts of rCTGF tocells treated with TGF-β and inhibited with either 8Br cAMP or choleratoxin overcomes the inhibition and allows the cells to grow at a ratecomparable to those treated with TGF-β in the absence of cAMP or choleratoxin (far right panel). These studies suggest a direct link between theproduction of CTGF and the ability of NRK cells to grow in suspension.They also demonstrate that while TGF-β can induce certain effects infibroblasts in the presence of elevated levels of cAMP they are notsufficient to allow for anchorage independent growth. Also, since CTGFalone is not sufficient to stimulate this biological response it is nota substitute for all of TGF-β's actions on fibroblasts. These resultsdemonstrate there are both CTGF dependent and CTGF independent effectsinduced by TGF-β in target cells (NRK) that act synergistically to allowfor a specific cellular response (anchorage independent growth).

EXAMPLE 13 Inhibition of TGF-β Induced Granulation Tissue Formation byAgents that Elevate cAMP Levels

TGF-β has been shown to induce fibrosis in several animal model studies.For example, one group injected 400 to 800 ng of TGF-β into thesubdermnal space in the back of neonatal mice. When the TGF-β isinjected once a day for three days in a row, a large area of fibrotictissue forms (Roberts, et al., Proc. Natl. Acad. Sci. USA, 83:4167,1986). The present example shows comparative studies with TGF-β and CTGFand the results showed that CTGF induced the formation of connectivetissue which is very similar, if not identical to that formed inresponse to TGF-β . Other growth factors such as PDGF or EGF do notinduce tissue similar to TGF-β, indicating that CTGF may be responsiblefor the formation of the tissue induced by TGF-β injection.

Because the results in Example 12 showed that cAMP levels could blockthe induction of CTGF in the cultured cells, it was of interest todetermine whether elevation of cAMP levels in cells in an animal couldblock the action of TGF-β in vivo. Using the injection model describedabove and in Roberts, et al., the following experiment was performed.Neonatal mice were injected once a day for three days in a row witheither: TGF-β (400 ng); cholera toxin (100 ng); TGF-β (400 ng) andcholera toxin (100 ng); or saline. Three mice were used in each group.After injected tissue was prepared using standard histological methods,the area of injection was examined by light microscopy after stainingwith hematoxylin and eosin.

As expected, saline injections had no effect on the type of tissuepresent in the murine skin and TGF-β injections induced a large amountof new connective tissue which resembled granulation tissue. This tissuecontained increased numbers of fibroblasts and increased amounts ofcollagen and other matrix components. Injection of cholera toxin alonecaused no stimulation of granulation tissue formation. Co-injection ofTGF-β and cholera toxin also showed no formation of granulation tissuedemonstrating that the cholera toxin blocked the TGF-β induced formationof granulation tissue. These results indicate the therapeutic utility ofagents that block the production or action of CTGF for use asanti-fibrotic drugs.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 9                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 2970                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo Sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (1025)...(2074)                                                - - <400> SEQUENCE: 1                                                         - - tctagagctc gcgcgagctc taatacgact cactataggg cgtcgactcg at -            #cccttttt     60                                                                 - - ctggaaacat tgatggccac tcgtcccttg tccttgccta tataaaactc ct -            #acatatat    120                                                                 - - taagagaaaa ctaagcaaga gttttggaaa tctgccccag gagactgcat cc -            #tgagtcac    180                                                                 - - acgagtcttt gttctctttc ttgtcccaaa accgttacct caagtgacaa at -            #gatcaaat    240                                                                 - - ctcaaatata gaattcaggg ttttacaggt aggcatcttg aggatttcaa at -            #ggttaaaa    300                                                                 - - gcaactcact ccttttctac tctttggaga gtttcaagag cctatagcct ct -            #aaaacgca    360                                                                 - - aatcattgct aagggttggg ggggagaaac cttttcgaat tttttaggaa tt -            #cctgctgt    420                                                                 - - ttgcctcttc agctacctac ttcctaaaaa ggatgtatgt cagtggacag aa -            #cagggcaa    480                                                                 - - acttattcga aaaagaaata agaaataatt gccagtgtgt ttataaatga ta -            #tgaatcag    540                                                                 - - gagtggtgcg aagaggatag gaaaaaaaaa ttctatttgg tgctggaaat ac -            #tgcgcttt    600                                                                 - - ttttttcctt tttttttttt tctgtgagct ggagtgtgcc agctttttca ga -            #cggaggaa    660                                                                 - - tgctgagtgt caaggggtca ggatcaatcc ggtgtgagtt gatgaggcag ga -            #aggtgggg    720                                                                 - - aggaatgcga ggaatgtccc tgtttgtgta ggactccatt cagctcattg gc -            #gagccgcg    780                                                                 - - gccgcccgga gcgtataaaa gcctcgggcc gcccgcccca aactcacaca ac -            #aactcttc    840                                                                 - - cccgctgaga ggagacagcc agtgcgactc caccctccag ctcgacggca gc -            #cgccccgg    900                                                                 - - ccgacagccc cgagacgaca gcccggcgcg tcccggtccc cacctccgac ca -            #ccgccagc    960                                                                 - - gctccaggcc ccgcgctccc cgctcgccgc caccgcgccc tccgctccgc cc -            #gcagtgcc   1020                                                                 - - aacc atg acc gcc gcc agt atg ggc ccc gtc c - #gc gtc gcc ttc gtg       gtc    1069                                                                          Met Thr Ala Ala Ser Met Gly Pro - # Val Arg Val Ala Phe Val Val               1            - #   5               - #    10              - #          15                                                                               - - ctc ctc gcc ctc tgc agc cgg ccg gcc gtc gg - #c cag aac tgc agc        ggg     1117                                                                    Leu Leu Ala Leu Cys Ser Arg Pro Ala Val Gl - #y Gln Asn Cys Ser Gly                           20 - #                 25 - #                 30              - - ccg tgc cgg tgc ccg gac gag ccg gcg ccg cg - #c tgc ccg gcg ggc gtg         1165                                                                       Pro Cys Arg Cys Pro Asp Glu Pro Ala Pro Ar - #g Cys Pro Ala Gly Val                        35     - #             40     - #             45                  - - agc ctc gtg ctg gac ggc tgc ggc tgc tgc cg - #c gtc tgc gcc aag cag         1213                                                                       Ser Leu Val Leu Asp Gly Cys Gly Cys Cys Ar - #g Val Cys Ala Lys Gln                    50         - #         55         - #         60                      - - ctg ggc gag ctg tgc acc gag cgc gac ccc tg - #c gac ccg cac aag ggc         1261                                                                       Leu Gly Glu Leu Cys Thr Glu Arg Asp Pro Cy - #s Asp Pro His Lys Gly                65             - #     70             - #     75                          - - ctc ttc tgt gac ttc ggc tcc ccg gcc aac cg - #c aag atc ggc gtg tgc         1309                                                                       Leu Phe Cys Asp Phe Gly Ser Pro Ala Asn Ar - #g Lys Ile Gly Val Cys            80                 - # 85                 - # 90                 - # 95       - - acc gcc aaa gat ggt gct ccc tgc atc ttc gg - #t ggt acg gtg tac cgc         1357                                                                       Thr Ala Lys Asp Gly Ala Pro Cys Ile Phe Gl - #y Gly Thr Val Tyr Arg                           100  - #               105  - #               110              - - agc gga gag tcc ttc cag agc agc tgc aag ta - #c cag tgc acg tgc ctg         1405                                                                       Ser Gly Glu Ser Phe Gln Ser Ser Cys Lys Ty - #r Gln Cys Thr Cys Leu                       115      - #           120      - #           125                  - - gac ggg gcg gtg ggc tgc atg ccc ctg tgc ag - #c atg gac gtt cgt ctg         1453                                                                       Asp Gly Ala Val Gly Cys Met Pro Leu Cys Se - #r Met Asp Val Arg Leu                   130          - #       135          - #       140                      - - ccc agc cct gac tgc ccc ttc ccg agg agg gt - #c aag ctg ccc ggg aaa         1501                                                                       Pro Ser Pro Asp Cys Pro Phe Pro Arg Arg Va - #l Lys Leu Pro Gly Lys               145              - #   150              - #   155                          - - tgc tgc gag gag tgg gtg tgt gac gag ccc aa - #g gac caa acc gtg gtt         1549                                                                       Cys Cys Glu Glu Trp Val Cys Asp Glu Pro Ly - #s Asp Gln Thr Val Val           160                 1 - #65                 1 - #70                 1 -      #75                                                                              - - ggg cct gcc ctc gcg gct tac cga ctg gaa ga - #c acg ttt ggc cca        gac     1597                                                                    Gly Pro Ala Leu Ala Ala Tyr Arg Leu Glu As - #p Thr Phe Gly Pro Asp                          180  - #               185  - #               190              - - cca act atg att aga gcc aac tgc ctg gtc ca - #g acc aca gag tgg agc         1645                                                                       Pro Thr Met Ile Arg Ala Asn Cys Leu Val Gl - #n Thr Thr Glu Trp Ser                       195      - #           200      - #           205                  - - gcc tgt tcc aag acc tgt ggg atg ggc atc tc - #c acc cgg gtt acc aat         1693                                                                       Ala Cys Ser Lys Thr Cys Gly Met Gly Ile Se - #r Thr Arg Val Thr Asn                   210          - #       215          - #       220                      - - gac aac gcc tcc tgc agg cta gag aag cag ag - #c cgc ctg tgc atg gtc         1741                                                                       Asp Asn Ala Ser Cys Arg Leu Glu Lys Gln Se - #r Arg Leu Cys Met Val               225              - #   230              - #   235                          - - agg cct tgc gaa gct gac ctg gaa gag aac at - #t aag aag ggc aaa aag         1789                                                                       Arg Pro Cys Glu Ala Asp Leu Glu Glu Asn Il - #e Lys Lys Gly Lys Lys           240                 2 - #45                 2 - #50                 2 -      #55                                                                              - - tgc atc cgt act ccc aaa atc tcc aag cct at - #c aag ttt gag ctt        tct     1837                                                                    Cys Ile Arg Thr Pro Lys Ile Ser Lys Pro Il - #e Lys Phe Glu Leu Ser                          260  - #               265  - #               270              - - ggc tgc acc agc atg aag aca tac cga gct aa - #a ttc tgt gga gta tgt         1885                                                                       Gly Cys Thr Ser Met Lys Thr Tyr Arg Ala Ly - #s Phe Cys Gly Val Cys                       275      - #           280      - #           285                  - - acc gac ggc cga tgc tgc acc ccc cac aga ac - #c acc acc ctg ccg gtg         1933                                                                       Thr Asp Gly Arg Cys Cys Thr Pro His Arg Th - #r Thr Thr Leu Pro Val                   290          - #       295          - #       300                      - - gag ttc aag tgc cct gac ggc gag gtc atg aa - #g aag aac atg atg ttc         1981                                                                       Glu Phe Lys Cys Pro Asp Gly Glu Val Met Ly - #s Lys Asn Met Met Phe               305              - #   310              - #   315                          - - atc aag acc tgt gcc tgc cat tac aac tgt cc - #c gga gac aat gac atc         2029                                                                       Ile Lys Thr Cys Ala Cys His Tyr Asn Cys Pr - #o Gly Asp Asn Asp Ile           320                 3 - #25                 3 - #30                 3 -      #35                                                                              - - ttt gaa tcg ctg tac tac agg aag atg tac gg - #a gac atg gca tga             2074                                                                      Phe Glu Ser Leu Tyr Tyr Arg Lys Met Tyr Gl - #y Asp Met Ala                                   340  - #               345                                     - - agccagagag tgagagacat taactcatta gactggaact tgaactgatt ca -             #catctcat   2134                                                                 - - ttttccgtaa aaatgatttc agtagcacaa gttatttaaa tctgtttttc ta -            #actggggg   2194                                                                 - - aaaagattcc cacccaattc aaaacattgt gccatgtcaa acaaatagtc ta -            #tcttcccc   2254                                                                 - - agacactggt ttgaagaatg ttaagacttg acagtggaac tacattagta ca -            #cagcacca   2314                                                                 - - gaatgtatat taaggtgtgg ctttaggagc agtgggaggg taccggcccg gt -            #tagtatca   2374                                                                 - - tcagatcgac tcttatacga gtaatatgcc tgctatttga agtgtaattg ag -            #aaggaaaa   2434                                                                 - - ttttagcgtg ctcactgacc tgcctgtagc cccagtgaca gctaggatgt gc -            #attctcca   2494                                                                 - - gccatcaaga gactgagtca agttgttcct taagtcagaa cagcagactc ag -            #ctctgaca   2554                                                                 - - ttctgattcg aatgacactg ttcaggaatc ggaatcctgt cgattagact gg -            #acagcttg   2614                                                                 - - tggcaagtga atttgcctgt aacaagccag attttttaaa atttatattg ta -            #aatattgt   2674                                                                 - - gtgtgtgtgt gtgtgtgtat atatatatat atatgtacag ttatctaagt ta -            #atttaaag   2734                                                                 - - ttgtttgtgc ctttttattt ttgtttttaa tgctttgata tttcaatgtt ag -            #cctcaatt   2794                                                                 - - tctgaacacc ataggtagaa tgtaaagctt gtctgatcgt tcaaagcatg aa -            #atggatac   2854                                                                 - - ttatatggaa attctgctca gatagaatga cagtccgtca aaacagattg tt -            #tgcaaagg   2914                                                                 - - ggaggcatca gtgtcttggc aggctgattt ctaggtagga aatgtggtag ct - #cacg           2970                                                                       - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 349                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo Sapiens                                                   - - <400> SEQUENCE: 2                                                         - - Met Thr Ala Ala Ser Met Gly Pro Val Arg Va - #l Ala Phe Val Val Leu       1               5  - #                10  - #                15               - - Leu Ala Leu Cys Ser Arg Pro Ala Val Gly Gl - #n Asn Cys Ser Gly Pro                  20      - #            25      - #            30                   - - Cys Arg Cys Pro Asp Glu Pro Ala Pro Arg Cy - #s Pro Ala Gly Val Ser              35          - #        40          - #        45                       - - Leu Val Leu Asp Gly Cys Gly Cys Cys Arg Va - #l Cys Ala Lys Gln Leu          50              - #    55              - #    60                           - - Gly Glu Leu Cys Thr Glu Arg Asp Pro Cys As - #p Pro His Lys Gly Leu      65                  - #70                  - #75                  - #80        - - Phe Cys Asp Phe Gly Ser Pro Ala Asn Arg Ly - #s Ile Gly Val Cys Thr                      85  - #                90  - #                95               - - Ala Lys Asp Gly Ala Pro Cys Ile Phe Gly Gl - #y Thr Val Tyr Arg Ser                  100      - #           105      - #           110                  - - Gly Glu Ser Phe Gln Ser Ser Cys Lys Tyr Gl - #n Cys Thr Cys Leu Asp              115          - #       120          - #       125                      - - Gly Ala Val Gly Cys Met Pro Leu Cys Ser Me - #t Asp Val Arg Leu Pro          130              - #   135              - #   140                          - - Ser Pro Asp Cys Pro Phe Pro Arg Arg Val Ly - #s Leu Pro Gly Lys Cys      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Cys Glu Glu Trp Val Cys Asp Glu Pro Lys As - #p Gln Thr Val Val        Gly                                                                                             165  - #               170  - #               175             - - Pro Ala Leu Ala Ala Tyr Arg Leu Glu Asp Th - #r Phe Gly Pro Asp Pro                  180      - #           185      - #           190                  - - Thr Met Ile Arg Ala Asn Cys Leu Val Gln Th - #r Thr Glu Trp Ser Ala              195          - #       200          - #       205                      - - Cys Ser Lys Thr Cys Gly Met Gly Ile Ser Th - #r Arg Val Thr Asn Asp          210              - #   215              - #   220                          - - Asn Ala Ser Cys Arg Leu Glu Lys Gln Ser Ar - #g Leu Cys Met Val Arg      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Pro Cys Glu Ala Asp Leu Glu Glu Asn Ile Ly - #s Lys Gly Lys Lys        Cys                                                                                             245  - #               250  - #               255             - - Ile Arg Thr Pro Lys Ile Ser Lys Pro Ile Ly - #s Phe Glu Leu Ser Gly                  260      - #           265      - #           270                  - - Cys Thr Ser Met Lys Thr Tyr Arg Ala Lys Ph - #e Cys Gly Val Cys Thr              275          - #       280          - #       285                      - - Asp Gly Arg Cys Cys Thr Pro His Arg Thr Th - #r Thr Leu Pro Val Glu          290              - #   295              - #   300                          - - Phe Lys Cys Pro Asp Gly Glu Val Met Lys Ly - #s Asn Met Met Phe Ile      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Lys Thr Cys Ala Cys His Tyr Asn Cys Pro Gl - #y Asp Asn Asp Ile        Phe                                                                                             325  - #               330  - #               335             - - Glu Ser Leu Tyr Tyr Arg Lys Met Tyr Gly As - #p Met Ala                              340      - #           345                                         - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 26                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR primer HO1                                        - - <400> SEQUENCE: 3                                                         - - cggaattcgc agtgccaacc atgacc          - #                  - #                  26                                                                      - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 26                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR primer H02                                        - - <400> SEQUENCE: 4                                                         - - ccgaattctt aatgtctctc actctc          - #                  - #                  26                                                                      - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 26                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR primer HO3                                        - - <400> SEQUENCE: 5                                                         - - cggaattcct ggaagacacg tttggc          - #                  - #                  26                                                                      - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 14                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: NF-1 like site                                       <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- difference                                        <222> LOCATION: (4)...(9)                                                     <223> OTHER INFORMATION: Each N can be A, G, - # T or C.                       - - <400> SEQUENCE: 6                                                         - - tggnnnnnng ccaa              - #                  - #                      - #     14                                                                   - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 10                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: TGF-beta inhibitory element- - #like sequence        <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- difference                                        <222> LOCATION: (2)...(3)                                                     <223> OTHER INFORMATION: Each N can be A, G, - # T or C.                       - - <400> SEQUENCE: 7                                                         - - gnnttggtga                - #                  - #                      - #        10                                                                   - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 13                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: TGF-beta responsive/regulatory - #element             - - <400> SEQUENCE: 8                                                         - - gtgtcaaggg gtc              - #                  - #                      - #      13                                                                   - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 838                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Mus musculus                                                   - - <400> SEQUENCE: 9                                                         - - tctttcttct cccactatat tccctgacac ttaggcttct gaagatagcc at -             #ttggtctg     60                                                                 - - aactcataaa cttatttttc tagaaaacca tgcccagtca taccccttgc ct -            #gcctggac    120                                                                 - - cctgaagaca agttcttaca taaagagtgc tgaaaatctt cctgggaacc ta -            #catccttg    180                                                                 - - gctttcatat ctttcagcca tcaaaatggc catctcagtg accaaagatc aa -            #tgcctgta    240                                                                 - - tttcagatac aaaagttgca cataggaatt ctgggaggag aggaggcatt tc -            #aaatggct    300                                                                 - - ataagcaccc ttctcctctc agtagaagaa caccaagaga ctacagcccc gt -            #aaagaaaa    360                                                                 - - aaaaaaaaaa atccaaaaca aagaaaaaga aatatttttt ttaatttcta gg -            #ggcccatg    420                                                                 - - gtatttgcct cttgagctat ttgagtcttg agaagttttt atgtcagtag cc -            #agaactgg    480                                                                 - - caaagagatt tttaagaaga aaagatcaga gaaataatcg tttatttcta ag -            #ttatattt    540                                                                 - - catcaggagg ggtgagaaga cgatatggag aaagttttac ttcttggtgt tg -            #tgctggaa    600                                                                 - - acacagcgcc tttttttttt ttttcctggc gagctaaagt gtgccagctt tt -            #tcagacgg    660                                                                 - - aggaatgtgg agtgtcaagg ggtcaggatc aatccggtgt gagttgatga gg -            #caggaagg    720                                                                 - - tggggaggaa tgtgaggaat gtccctgttt gtgtaggact tcattcagtt ct -            #ttggcgag    780                                                                 - - ccggctcccg ggagcgtata aaagccagcg ccgcccgcct agtctcacac ag -            #ctcttc      838                                                              __________________________________________________________________________

We claim:
 1. A method for identifying a composition which affects TGF-βinduced connective tissue growth factor (CTGF) expression, the methodcomprising:(a) incubating components comprising the composition, TGF-β,and a nucleic acid molecule comprising an expressible nucleotidesequence operably linked to a TGF-β regulatory element (TβRE),said TβREcomprising the nucleotide sequence 5'-GTGTCAAGGGGTC-3' (SEQ ID NO: 8),wherein the incubating is carried out under conditions and for a timesufficient to allow the components to interact; and (b) comparingexpression of the expressible nucleotide sequence operably linked to theTβRE in the presence of the composition with the level of expression ofthe nucleotide sequence in the absence of the composition, wherein adifference in the levels of expression identifies a composition thataffects TGF-β induced CTGF expression.
 2. The method of claim 1, whereinthe composition inhibits CTGF expression.
 3. The method of claim 1,wherein the composition stimulates CTGF expression.
 4. The method ofclaim 1, wherein the expressible nucleotide sequence is selected fromthe group consisting of β-lactamase, chloramphenicol acetyltransferase(CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase(neo, G418), dihydrofolate reductase (DHFR),hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK),β-galactosidase (β-gal), and xanthine guanine phosphoribosyltransferse(XGPRT).
 5. The method of claim 1, wherein said TβRE comprisesnucleotides 664 to 698 as set forth in SEQ ID NO:
 1. 6. The method ofclaim 1, wherein said TβRE comprises nucleotides 1 to 897 as set forthin SEQ ID NO:
 1. 7. A method of identifying a composition that regulatesconnective tissue growth factor (CTGF) expression from a TGF-βregulatory element (TβRE), the method comprising:(a) incubating anucleic acid molecule comprising a TβRE comprising the nucleotidesequence 5'-GTGTCAAGGGGTC-3' (SEQ ID NO: 8) operably linked to anexpressible nucleotide sequence, and a composition suspected of havingthe ability to regulate expression of the expressible nucleotidesequence from the TβRE, under conditions and for a time sufficient toallow such expression; and (b) comparing the level of expression of theexpressible nucleotide sequence in the presence of the composition withthe level of expression in the absence of the composition, wherein adifference in the level of expression identifies a composition thatregulates CTGF expression from the TβRE.
 8. The method of claim 7,wherein said TβRE comprises nucleotides 664 to 698 as set forth in SEQID NO:
 1. 9. The method of claim 7, wherein said TβRE comprisesnucleotides 1 to 897 as set forth in SEQ ID NO: 1.